Purified and isolated piwi family genes and gene products and methods using same

ABSTRACT

A family of isolated and purified proteins and nucleic acids are disclosed. Particularly, piwi family proteins and cDNAs encoding the same are disclosed. Recombinant host cells, recombinant nucleic acids, recombinant proteins and transgenic animals are also disclosed, along with methods of producing each. Isolated and purified antibodies to piwi family homologs, and methods of producing the same, are also disclosed. piwi family gene products are characterized as having activity in the growth, proliferation and self-renewing division of stem cells, and proliferation of primordial germ cells. Thus, therapeutic, screening, culturing and transgenic methods involving these activities are also disclosed.

PRIORITY APPLICATION INFORMATION

[0001] This application is a continuation-in-part of InternationalApplication No.

[0002] PCT/US99/28764, filed Dec. 3, 1999, which claims the benefit ofU.S. Provisional Patent Application 60/110,091, filed Dec. 4, 1998, nowabandoned. The disclosure of International Application No.PCT/US99/28764 and U.S. Provisional Patent Application 60/110,091 areeach incorporated herein by reference in their entirety.

GRANT STATEMENT

[0003] This work was supported by NIH grant HD 33760. The U.S.

[0004] Government has certain rights in the invention.

TECHNICAL FIELD

[0005] The present invention relates generally to isolated and purifiedproteins and nucleic acids which modulate stem cell renewal, growth anddivision and which modulate primordial germ cell proliferation. Moreparticularly, the present invention relates to isolated and purifiedpiwi family proteins and isolated and purified polynucleic acidsencoding the same.

[0006] The publications and other materials used herein to illuminatethe background of the invention, and in particular cases, to provideadditional details respecting the practice, are incorporated herein byreference, and for convenience, are referenced by author and year ofpublication in the following text, and respectively grouped by author inthe appended list of references. Table of Abbreviations APC apical polarcell(s) ATCC American Type Culture Collection bFGF basic fibroblastgrowth factor BRL buffalo rat liver BSA bovine serum albumin Cbcystoblast CDR(s) complementarity determining region(s) COOH carboxyterminus CyO a strain of Drosophila DAPI a DNA specific dye DMEMDulbecco's modified Eagle's medium DC differentiating 16-cell cysts dpcdays post coitum dpp days post partum DTC distal tip cell ECL enhancedchemiluminescent EDTA ethylenediamine tetraacetate ES epithetial sheathEST expressed sequence tags FBS fetal bovine serum FITC fluoresceinisothiocyanate FC follicle cell FRT^(40A) a strain of Drosophila Fufusome GC-MS gas chromatography-mass spectroscopy Ge germarium(ia) GFPgreen fluorescent protein GSC(s) germline stem cell(s) HAT cell culturemedia comprising hypoxanthine, aminopterin, and thymidine hiwi/HIWI ahuman homolog of the piwi/PIWI gene and gene product HPLC high pressureliquid chromatography hsGal4 a heat shock inducible transgene IS innersheath KLH keyhole limpet hemocyanin MC mitotically active cystsmiwi/MIWI a murine homolog of the piwi/PIWI gene and gene product MmGFPmutant GFP with improved stability MPZ mitotic proliferation zone mychuman oncogene used herein as molecular tag for piwi NH2 amino terminusORF open reading frame PCR polymerase chain reaction PGC(s) primordialgerm cell(s) piwi/PIWI a gene and gene product isolated from Drosophilaand having stem cell division modulating activity RACE rapidamplification of cDNA ends RNAi RNA interference assay SSC(s) somaticstem cell(s) Sp spectrosome TFC terminal filament cells TSXtestis-specific X-linked gene product TUNEL TdT-mediateddUTP-digoxigenin nick end- labeling UTR untranslated region VASA agermline specific marker WT wild type

BACKGROUND ART

[0007] Stem cells are a very small number of founder cells that play acentral role in tissue development and maintenance. In human bodies,stem cells are responsible for generating and/or maintainingapproximately 90% of cells in the adult tissues. Over-proliferation ofmalignant stem cells is the leading cause of cancer whileunder-proliferation of stem cells or stem-like progenitor cells leads totissue dystrophy, anemia, immunodeficiency, and male infertility. Thecrucial role of stem cells has long been attributed to their ability toself-renew and to generate immense number of specialized cells ondemand.

[0008] The ability of stem cells to self-renew and to produce a largenumber of differentiated progeny is critical for the development andmaintenance of a wide variety of tissues in organisms ranging frominsects to mammals (reviewed in Potten, 1997; Lin, 1997; Lin andSchagat, 1997; Morrison et al., 1997). This self-renewing ability iscontrolled both by extrinsic signaling and by cell-autonomous mechanisms(reviewed in Morrison et al., 1997; Lin and Schagat, 1997). Cellautonomous mechanisms have been elucidated in a few stem cell modelssuch as neuroblasts and germline stem cells in Drosophila (Lin andSchagat, 1997; Deng and Lin, 1997), whereas the role of extrinsicsignaling has been elucidated in several systems. For example, theproliferation and differentiation of mammalian stem cells in thehematopoietic, epidermal, and nervous systems depend on extrinsicsignals that act on specific receptors on the stem cell surface(Morrison et al., 1997).

[0009] In diverse organisms ranging from invertebrates to mammals, theproliferation of germ cells, some of which possess stem cell properties,has been postulated, and, in some cases, shown to be regulated byneighboring non-mitotic somatic cells (Lin, 1997). Particularly, in C.elegans, cell-cell interactions between the somatic distal tip cell(DTC) at the end of each gonadal arm and the underlying mitotic germlinenuclei via the lag-2lglp-1 signaling pathway provides a paradigm forsoma-germline interaction (reviewed in Kimble and Simpson, 1997). Theglp-1 pathway is required to maintain a population of mitotically activenuclei in the germline.

[0010] However, few molecules and/or mechanisms identified in aparticular type of stem cells have been shown to be applicable to otherstem cell systems. For example, the glp-1 equivalent pathway inDrosophila does not play a role in regulating GSC division andmaintenance (Ruohala et al., 1991; Xu et al., 1992).

[0011] The self-renewing asymmetric division of GSCs in the Drosophilaovary is known to be controlled both by an intracellular mechanism (Dengand Lin, 1997) and by cell-cell interactions (Lin and Spradling, 1993).The intracellular mechanism involves a cytoplasmic organelle termed thespectrosome that controls the orientation of GSC division (Lin et al.,1994; Deng and Lin, 1997). The cell-cell interaction mechanism involvesterminal filament cells, as shown by laser ablation studies (Lin andSpradling, 1993). Recently, dpp has been shown as a key signalingmolecule required for GSC division and maintenance (Xie and. Spradling,1998). It is possible that the dpp signal emanates from somatic cells.Alternatively, dpp signal may originate from the germline or even withinGSCs, like its mammalian homologs (Zhao et al., 1996).

[0012] In mammals, primordial germ cells cultured from the genital ridgehave the ability to give rise to pluripotent embryonic stem cells. Forexample, U.S. Pat. No. 5,690,926 issued Nov. 25, 1997 to Hogan; U.S.Pat. No. 5,670,372 issued Sep. 23, 1997 to Hogan; and U.S. Pat. No.5,537,357 issued Sep. 26, 1995 to Hogan each disclose pluripotentialmammalian embryonic stem cells and methods of making the same. Thedisclosure of these patents is limited to mammalian embryonic stem cellsand particularly to the culturing of murine and other mammalianembryonic stem cells using a combination of growth factors consisting ofSCF, FGF and LIF.

[0013] Current prior art reports on the culture of avian primordial germcells (PGCS) have concentrated on efforts to maintain a PGC-phenotypeand to stimulate proliferation. See e.g., Chang. I. K. et al., Cell.Biol. Int. 1997 August; 21(8): 495-9; Chang, I. K. et al., Cell. Biol.Int. 1995 February; 19(2): 143-9; Alloli, N. et al., Dev. Biol. 1994September; 165(1): 30-7 and PCT Publication No. WO 99/06533, publishedFeb. 11, 1999, (Applicant—University of Massachusetts; Inventors—Poncede Leon et al.).

[0014] As illustrated above, numerous attempts have been devoted toidentify genes that control the self-renewing ability of stem cells orthe proliferation of primordial germ cells. As a result, a number ofgrowth factors and signaling molecules, such as Steel factor and itsc-kit receptor, have been identified to regulate such activity incertain tissues. Despite this progress, there remains a long-felt andcontinuing need to identify genes that play a role in modulating thegrowth and self-renewing division of stem cells, particularly GSCs, andthat play a role in modulating proliferation of primordial germ cells.

SUMMARY OF THE INVENTION

[0015] The present invention contemplates an isolated and purifiedfamily of genes and gene products (the piwi family) which plays a rolein the growth, proliferation and self-renewing division of stem cells,and proliferation of primordial germ cells. More preferably, apolypeptide of the invention is a recombinant polypeptide. Even morepreferably, a polypeptide of the present invention comprises avertebrate piwi family polypeptide. Even more preferably, a polypeptideof the present invention comprises a mammalian piwi family polypeptide.Even more preferably, a polypeptide of the present invention comprises ahuman piwi family polypeptide. Even more preferably, a polypeptide ofthe present invention comprises an amino acid sequence from the aminoacid residue sequences of any of SEQ ID NOs:2, 4 and 6.

[0016] The present invention also provides an isolated and purifiedpolynucleotide that encodes a polypeptide that plays a role in thegrowth, proliferation and self-renewing division of stem cells, andproliferation of primordial germ cells. In a preferred embodiment, apolynucleotide of the present invention comprises a DNA molecule from avertebrate species. Preferred vertebrates comprise mammals, birds orfish. A preferred mammal is a human. More preferably, a polynucleotideof the present invention encodes a polypeptide designated PIWI. Evenmore preferably, a polynucleotide of the present invention encodes apolypeptide comprising an amino acid residue sequence of any of SEQ IDNOs:2, 4 and 6. Most preferably, an isolated and purified polynucleotideof the invention comprises a nucleotide base sequence of any of SEQ IDNOs:1, 3 and 5.

[0017] In another embodiment, the present invention contemplates anantibody immunoreactive with a piwi family polypeptide as describedabove. SEQ ID NOs:3-6 set forth nucleotide and amino acid sequences fromrepresentative vertebrates, human and mouse. Also contemplated by thepresent invention are antibodies immunoreactive with homologues orbiologically equivalent piwi family polynucleotides and polypeptidesfound in other vertebrates. Preferably, an antibody of the invention isa monoclonal antibody. More preferably, the piwi family polypeptidecomprises human PIWI (HIWI). Even more preferably, the piwi polypeptidecomprises an amino acid residue sequence of any of SEQ ID NOs:2, 4 and6.

[0018] In another aspect, the present invention contemplates a method ofproducing an antibody immunoreactive with a piwi family polypeptide asdescribed above, the method comprising the steps of (a) transfecting arecombinant host cell with a polynucleotide that encodes a biologicallyactive piwi family polypeptide; (b) culturing the host cell underconditions sufficient for expression of the polypeptide; (c) recoveringthe polypeptide; and (d) preparing the antibody to the polypeptide. SEQID NOs:3-6 set forth nucleotide and amino acid sequences fromrepresentative vertebrates, human and mouse. Preferably, the host cellis transfected with a polynucleotide of any of SEQ ID NOs:1, 3 and 5.Even more preferably, the present invention provides an antibodyprepared according to the method described above. Also contemplated bythe present invention is the use of homologues or biologicallyequivalent polynucleotides and polypeptides found in other vertebratesto produce antibodies.

[0019] Alternatively, the present invention provides a method ofdetecting a piwi family polypeptide as described above, wherein themethod comprises immunoreacting the polypeptide with an antibodyprepared according to the method described above to form anantibody-polypeptide conjugate, and detecting the conjugate.

[0020] In yet another embodiment, the present invention contemplates amethod of detecting a messenger RNA transcript that encodes a piwifamily polypeptide as described above, wherein the method compriseshybridizing the messenger RNA transcript with a polynucleotide sequencethat encodes that polypeptide to form a duplex; and detecting theduplex. Alternatively, the present invention provides a method ofdetecting a DNA molecule that encodes a piwi family polypeptide asdescribed above, wherein the method comprises hybridizing DNA moleculeswith a polynucleotide that encodes a biologically active piwi familypolypeptide to form a duplex; and detecting the duplex.

[0021] In another aspect, the present invention contemplates an assaykit for detecting the presence of a piwi family polypeptide in abiological sample, where the kit comprises a first container containinga first antibody capable of immunoreacting with a biologically activepiwi polypeptide, with the first antibody. Preferably, the firstantibody is present in an amount sufficient to perform at least oneassay. Also preferably, an assay kit of the invention further comprisesa second container containing a second antibody that immunoreacts withthe first antibody. More preferably, the antibodies used in an assay kitof the present invention are monoclonal antibodies. Even morepreferably, the first antibody is affixed to a solid support. Morepreferably still, the first and second antibodies comprise an indicator,and, preferably, the indicator is a radioactive label, a fluorescentlabel or an enzyme.

[0022] In an alternative aspect, the present invention provides an assaykit for detecting the presence, in biological samples, of a piwi familypolypeptide, the kits comprising a first container that contains asecond polynucleotide identical or complementary to a segment of atleast 10 contiguous nucleotide bases of a polynucleotide that encodes abiologically active piwi family polypeptide.

[0023] In another embodiment, the present invention contemplates anassay kit for detecting the presence, in a biological sample, of anantibody immunoreactive with a piwi family polypeptide, the kitcomprising a first container containing a biologically active piwifamily polypeptide that immunoreacts with the antibody, with thepolypeptide present in an amount sufficient to perform at least oneassay.

[0024] In still a further embodiment, this invention pertains totransgenic, therapeutic, screening and culturing methods based upon theself-renewing division of stem cells, and proliferation of primordialgerm cells function of piwi family polynucleotides and polypeptides asdescribed herein. Representative therapeutic and culturing methodsinclude administration of a soluble form of the piwi protein and genetherapy approaches using an isolated and purified polynucleotide of thepresent invention.

[0025] Thus, a key aspect of this invention pertains to the discovery ofthe novel piwi family of proteins and nucleic acids. Preferred nucleicacid and amino acid sequences are described in SEQ ID NOs:1-6.

[0026] It is thus another aspect of this invention to provide a purifiedand isolated piwi family polypeptide having a role in the growth,proliferation and/or self-renewing division of stem cells, andproliferation of primordial germ cells.

[0027] The foregoing aspects and embodiments have broad utility giventhe biological significance of stem cell growth, proliferation andrenewal, and primordial germ cell proliferation. By way of example, theforegoing aspects and embodiments are useful in the preparation ofscreening assays and assay kits that are used to identify compounds thataffect or modulate piwi family biological activity, or that are used todetect the presence of the proteins and nucleic acids of this inventionin biological samples. Additionally, it is well known that isolated andpurified polypeptides have utility as feed additives for livestock andfurther polynucleotides encoding the polypeptides are thus useful inproducing the polypeptides.

[0028] Some of the aspects and objects of the invention having beenstated hereinabove, other aspects and objects will become evident as thedescription proceeds, when taken in connection with the accompanyingExamples and Drawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1 is a molecular map of the piwi locus. Restriction enzymemap for 15 kb of genomic DNA surrounding the piwi mutations is shown.Three transcripts are represented by thick arrows indicating theirrelative positions, sizes, splice sites and direction of transcription.The relative insertion sites of the P-element alleles is indicated abovethe restriction map with orientation of insertion indicated by black(5′) and white (3′) boxes. The P insertions are not shown according tothe scale, with piwi² insertion partially mapped. Restriction sites: R,EcoRI; H, HindIII; P, PstI; S, SalI; X, XhoI.

[0030]FIG. 2 depicts alignment of predicted PIWI protein sequence withits homologs in the piwi box domain in 22 different proteins. Thealignments were generated using Block Maker™ analysis (Henikoff et al.,1995) against amino acids 740-782 of the predicted piwi sequence. Theamino acid sequences were deduced from genomic DNA sequences for C.elegans (Ce G) or from cDNA (EST) sequences (E) for Rattus sp. (Rs) andOryza sativa (Os). GenBank accession numbers and cosmid designations(Ce) are indicated for each sequence. Residues identical to theconsensus are highlighted. Shown below the alignments, residues whichare absolutely conserved are shown as capital letters and residues whichare 80% or more conserved are shown as lower case letters while dashesindicate their spacing.

[0031]FIG. 3 is a graph showing that prg-RNAi significantly reducesgermline proliferation in C. elegans and that reducing prg activityaffects fertility. The graph indicates the number of progeny produced bya single F1 worm from an RNAi prg-1 or pBST injected mother vs. thepercentage of F1 animals which produced that given number of progeny.Comparison of prg-RNAi (striped bars) with pBST RNAi (solid bars)animals reveals two distinctive distributions.

[0032]FIG. 4 depicts the construction of full length hiwi cDNA clone.The 2.3 kb cDNA fragment obtained from library screening (herein named3-12) was subcloned into a pBluescript® KS⁺ plasmid vector, the 3-12insert is shown as an open box, the polycloning site on the vector isshown as a dark line. The 1.4 kb cDNA fragment obtained from 5′RACE(herein named 1501) was subcloned into a pGEM-T Easy™ vector. The 1501in pGEM-T Easy™ vector was digested with SacI to construct a vector forsubsequent ligation. The 3-12 in pBluescript® KS⁺ vector was partiallydigested with SacI to obtain the insert. This partially digested 3-12insert DNA was used to do ligation with 1501 in pGEM-T easy vector toconstruct a full length hiwi cDNA clone.

[0033]FIGS. 5A-5D is a schematic depicting the miwi locus (genomiclocation and gene structure).

[0034]FIG. 5A is a schematic depicting mouse chromosome 5.

[0035]FIG. 5B is a schematic depicting the miwi genomic locus.

[0036]FIG. 5C is a schematic depicting the location and structure on themiwi cDNA.

[0037]FIG. 5D is a schematic depicting the location and structure ofmiwi genomic clones.

[0038]FIG. 6 depicts an alignment of MIWI protein sequence with HIWI,Drosophila PIWI and C. elegans PRG-1 and PRG-2. Sequences names areshown on the right, and the position of each sequence is shown on theleft. Amino acid residues identical among above four genes are boxed inblack. The highly conserved C-terminal 104 amino acid residues(including the PIWI box) are underlined.

[0039]FIG. 7 is a schematic diagram of a Drosophila germarium. Theschematized germanium is oriented with the apical end toward the left.The numbers below the germarium in the brackets denote distinct regionswithin the germarium. Germarial region 1—extends from the apicalterminal filament cells (TFC, including cap cells, CpC) to the zone ofmitotically active cysts. Germarial regions 2a and 2b includedifferentiating 16-cell cysts. Two somatic stem cells (SSC) are locatedat the border between regions 2a and 2b. Germarial region 3 contains anewly formed egg chamber that is preparing to bud off from the germariumand contribute to the developing string of egg chambers that comprisethe rest of the ovariole. Abbreviations: GSC, germline stem cell; Sp,spectrosome; ISC, inner sheath cell; Cb, cystoblast; Fu, fusome; MC,mitotically active cysts; DC, differentiating 16-cell cysts; FC,follicle cell.

[0040]FIG. 8 is a schematic diagram of the myc-PIWI and PIWI-mycconstructs. The myc-piwi transgene encodes a 97.5 kDa protein. In themyc-PIWI construct, the myc epitope is inserted after the third aminoacid residue of PIWI. In the PIWI-myc construct, the epitope isinserted. 13 amino acids downstream of the highly conserved PIWI box(hatched), 48 residues away from the C-terminus. Abbreviations: NH2, theamino terminus; COOH, the carboxy terminus.

[0041]FIG. 9 is a graph displaying the relative division rates ofwild-type and piwi-GSCs and demonstrating that PIWI actscell-autonomously in GSCs to promote their division. The number ofgermaria counted is given in parentheses.

[0042]FIG. 10 is a graph displaying a comparison of the number ofspectrosomes present per wild-type germarium versus perpiwi-overexpressed germarium and demonstrating that somatic PIWIover-expression increases the number of germline stem cells. The numberof germaria examined (n) is in parentheses.

[0043]FIGS. 11A-11D are a summary of the targeting strategy and knockoutof the miwi gene.

[0044]FIG. 11A is a schematic diagram depicting the genomic organizationand targeting of miwi. The GFP reporter (hatched rectangle) and theloxP[PGK-neo] cassette (open rectangle) replace a 15.5 kb ApaI-KpnIgenomic region spanning exons 3-22. Exons are represented by filledrectangles (black), and exons 1, 2, 3, and 22 are indicated with theappropriate number designation below the corresponding filled rectangle.The flanking genomic regions used in the targeting vector are shown bythicker lines. The flanking genomic regions used in the targeting vectorare shown by thicker lines. ATG, translation start methionine; TAA,translation stop; A, ApaI; E, EcoRI; H, HindIII; K, KpnI; S, SacI; X,XbaI. Shaded rectangles (gray) labeled “5′probe” and “3′probe” indicatesthe positions probe sequences used for Southern blot analysis presentedin FIG. 11B. Arrows labeled “a”, “b”, and “c” identify the positions ofprimer sequences used for genotyping analysis presented in FIG. 11C.Scale bar denotes 1-kb.

[0045]FIG. 11B presents autoradiographs of two Southern-blots preparedwith genomic DNA from wildtype (MIWI^(+/−)), heterozygous (MIWI^(+/−))and homozygous (MIWI^(−/−)) mice and probed with 5′ and 3′ probes(indicated in FIG. 11A). For 5′ probe analysis, genomic DNA was digestedby HindIII; the wildtype allele is 6 kb whereas the targeted allele is4.8 kb. For 3′ probe analysis, genomic DNA was digested by EcoRI; thewildtype allele is 24.6 kb whereas the targeted allele is 10.2 kb.

[0046]FIG. 11C is a photograph of a gel showing PCR genotyping analysis.DNA prepared from wildtype, heterozygous miwi^(+/−), and homozygousmiwi^(−/−) mice was amplified using primers a, b and c (indicated inFIG. 11A). The wildtype allele yields a 0.64 kb PCR product, whereas thetargeted allele yields a 0.39 kb PCR product. Sizes (in kb) of molecularweight markers are indicated at left.

[0047]FIG. 11D presents a Western blot and corresponding Coomassieblue-stained gel. The Western blot was prepared using testicularextracts from wildtype, heterozygous miwi^(+/−), and homozygousmiwi^(−/−) mice, and the MIWI protein was detected using an anti-MIWIantibody. MIWI protein is absent from miwi^(−/−) mice. The duplicateSDS-PAGE gel stained by Coomassie blue shows equal loading of thetesticular extracts in all lanes. Sizes of protein markers (in kd) areindicated at left.

[0048]FIGS. 12A-12B present the targeting strategy and knockout of thesequence encoding the C-terminal region of MIWI.

[0049]FIG. 12A is a schematic diagram of miwi^(+/+) (miwi⁺, wild type)and miwi^(ΔC) alleles. The GFP reporter (hatched rectangle) and theloxP[PGK-neo] cassette (open rectangle) replace the SacI-KpnI genomicregion spanning exons 16-22, which encodes the last 245 amino acidresidues (including the last 6 residues encoded by exon 16) and 491 basepairs of 3′ untranslated region. Exons are represented by filledrectangles, and exons 1, 16, and 22 are indicated with the appropriatenumber designation below the corresponding filled rectangle. Theflanking genomic regions used in the targeting vector are shown bythicker lines. ATG, translation start methionine; TAA translation stop;TAG, translation stop; A, ApaI; E, EcoRI; H, HindIII; K, KpnI; S, SacI;X, XbaI. Scale bar denotes 1-kb.

[0050]FIG. 12B is a Western blot (lanes 1-5) and a Coomassieblue-stained duplicate SDS-PAGE gel (lanes 1′-5′) of testicular extractsfrom miwi^(null)/miwi^(null) (lanes 1 and 1′), miwi^(null/+)(lanes 2 and2′), +/+(lanes 3 and 3′), miwi^(ΔC/+) (lanes 4 and 4′), andmiwi^(ΔC)/miwi^(ΔC) (lanes 5 and 5′) mice. MIWI protein was detectedusing the MIWI34 anti-MIWI antibody. The truncated MIWI protein ispresent in homozygous miwi^(ΔC) mice (lane 5) and retains a molecularweight similar to the wildtype MIWI protein because of the MmGFP fusion.Lanes 1′-5′ demonstrate equal protein loading for lanes 1-5.

[0051]FIG. 13 presents graphs depicting the percentage of hematopoieticcells derived bone marrow surviving at the 2, 4, or. 6 weeks followingbone marrow transplantation (Wks post BMT) into irradiated ly5.1 mice.The left graph, labeled above as “1:1” shows the results oftransplantation experiments wherein a 1:1 ratio of miwi^(+/−) andmiwi^(−/−) (marked by ly5.2) was used. The right graph, labeled above as“2:1” shows the results of transplantation experiments wherein a 2:1ration of miwi^(+/−) and miwi^(−/−) (marked by ly5.2) were used. (▪),hematopoietic cells derived from bone marrow of miwi^(+/−) mice; (□),hematopoietic cells derived from bone marrow of miwi^(−/−) mice.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention discloses a novel class of genes requiredfor the growth, proliferation and self-renewing division of stem cells,and proliferation of primordial germ cells. The present inventionrelates to the discovery of a novel class of evolutionarily conservedgenes, the piwi family genes, that encode a novel group of basicproteins important for stem cell division, primordial germ cellproliferation and other developmental processes.

[0053] The disclosure of the present invention demonstrates that PIWIprotein can localize either in the nucleoplasm or cytoplasm and that thedose of the PIWI protein determines both the number and the mitotic rateof germline stem cells, and that the PIWI protein is localized in thegermplasm (the cytoplasm of future germ cells) and plays a key role indetermining the initial number of germ cells in during embryogenesis.The mouse PIWI protein (i.e., MIWI protein) is also a cytoplasmic factorwith very similar functions in germ cell and stem cell determination.

[0054] To identify genes essential for stem cell division, germline stemcells in the Drosophila ovary were established as a model by directlyidentifying these cells and demonstrating their self-renewing asymmetricdivision pattern. This stem cell model allowed applicant to effectivelycircumvent difficulties intrinsic to studying mammalian stem cells, suchas their rarity, lack of unique morphological markers, hyper-sensitivityto in vitro manipulation, and inaccessibility to systematic geneticscreen. Systematic genetic screens were then conducted to identifymutations that either abolish the self-renewing ability of stem cells orthe asymmetry of stem cell division, or both. These screens led to theidentification of the Drosophila piwi gene that is essential forgermline stem cell self-renewal. Subsequent cloning and molecularanalysis of the piwi gene and its homologs in C. elegans, the mouse, andhuman, and the identification of piwi-like genes in Arabidopsis led tothe following novel apsects of the present invention.

Definitions and Techniques Affecting Gene Products and Genes

[0055] As used in the following detailed description and in the claims,the term “piwi family” refers to a family or group of genes and geneproducts including, but not limited to, PIWI, HIWI, MIWI, PRG-1 andPRG-2 proteins, and the piwi, hiwi, miwi, prg-1 and prg-2 genes, each ofwhich are further defined herein. The term “piwi family” also includesother members of the piwi family of genes and gene productscharacterized by biological activity, including but not limited to thebiological activities of modulating growth, proliferation and/orself-renewing division of stem cells, and/or proliferation of primordialgerm cells.

[0056] Preferably, piwi family genes and gene products are isolated fromeukaryotic sources. Thus, the term “piwi family” also includesinvertebrate homologs. The term “piwi family” further includesvertebrate homologs of piwi family members, including, but not limitedto, mammalian, avian and fish homologs. Preferred mammalian homologs ofpiwi family members include, but are not limited to, murine and humanhomologs.

[0057] The terms “piwi family gene product”, “piwi family protein” and“piwi family polypeptide” refer to proteins having amino acid sequenceswhich are substantially identical to the native amino acid sequences inthe piwi family and which are biologically active in that they arecapable of modulating growth, proliferation and/or self-renewingdivision of stem cells, and/or proliferation of primordial germ cells,or cross-reacting with anti-piwi family antibodies raised against a piwifamily polypeptide.

[0058] The terms “piwi family gene product”, “piwi family protein” and“piwi family polypeptide” also include analogs of piwi family moleculeswhich exhibit at least some biological activity in common with nativepiwi family gene products. Furthermore, those skilled in the art ofmutagenesis will appreciate that other analogs, as yet undisclosed orundiscovered, may be used to construct piwi family analogs. There is noneed for an “piwi family gene product”, “piwi family protein” or “piwifamily polypeptide” to comprise all, or substantially all of the aminoacid sequence of a native piwi family gene product. Shorter or longersequences are anticipated to be of use in the invention. Thus, the term“piwi family gene product” also includes fusion or recombinant piwifamily polypeptides and proteins. Methods of preparing such proteins aredescribed herein in the Examples, among other places.

[0059] The terms “piwi family gene”, “piwi family gene sequence” and“piwi family gene segment” refer to any DNA sequence that issubstantially identical to a polynucleotide sequence encoding a piwifamily gene product, piwi family protein or piwi family polypeptide asdefined above. The terms also refer to RNA, or antisense sequences,compatible with such DNA sequences. A “piwi family gene”, “piwi familygene sequence” and “piwi family gene segment” may also comprise anycombination of associated control sequences.

[0060] The term “substantially identical”, when used to define either apiwi family gene product or piwi family amino acid sequence, or a piwifamily gene or piwi family nucleic acid sequence, means that aparticular sequence, for example, a mutant sequence, varies from thesequence of a natural piwi family by one or more deletions,substitutions, or additions, the net effect of which is to retain atleast some of biological activity of piwi family. Alternatively, DNAanalog sequences are “substantially identical” to specific DNA sequencesdisclosed herein if: (a) the DNA analog sequence is derived from codingregions of the natural piwi family gene; or (b) the DNA analog sequenceis capable of hybridization of DNA sequences of (a) under moderatelystringent conditions and which encode biologically active piwi familygene product; or (c) the DNA sequences are degenerative as a result ofthe genetic code to the DNA analog sequences defined in (a) and/or (b).Substantially identical analog proteins will be greater than about 60%identical to the corresponding sequence of the native protein. Sequenceshaving lesser degrees of similarity but comparable biological activityare considered to be equivalents. In determining nucleic acid sequences,all subject nucleic acid sequences capable of encoding substantiallysimilar amino acid sequences are considered to be substantially similarto a reference nucleic acid sequence, regardless of differences in codonsequences.

Percent Similarity

[0061] Percent similarity may be determined, for example, by comparingsequence information using the GAP computer program, available from theUniversity of Wisconsin Geneticist Computer Group. The GAP programutilizes the alignment method of Needleman et al. 1970, as revised bySmith et al. 1981. Briefly, the GAP program defines similarity as thenumber of aligned symbols (i.e. nucleotides or amino acids) which aresimilar, divided by the total number of symbols in the shorter of thetwo sequences. The preferred default parameters for the GAP programinclude: (1) a unitary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) of nucleotides and the weightedcomparison matrix of Gribskov et al., 1986, as described by Schwartz etal., 1979; (2) a penalty of 3.0 for each gap and an additional 0.01penalty for each symbol and each gap; and (3) no penalty for end gaps.Other comparison techniques are described in the Examples.

[0062] The term “homology” describes a mathematically based comparisonof sequence similarities which is used to identify genes or proteinswith similar functions or motifs. Accordingly, the term “homology” issynonymous with the term “similarity” and “percent similarity” asdefined above. Thus, the phrases “substantial homology” or “substantialsimilarity” have similar meanings.

Nucleic Acid Sequences

[0063] In certain embodiments, the invention concerns the use of piwifamily genes and gene products that include within their respectivesequences a sequence which is essentially that of a piwi family gene, orthe corresponding protein. The term “a sequence essentially as that of apiwi family gene”, means that the sequence substantially corresponds toa portion of a piwi family polypeptide or piwi family gene and hasrelatively few bases or amino acids (whether DNA or protein) which arenot identical to those of a piwi family protein or piwi family gene, (ora biologically functional equivalent of, when referring to proteins).The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequenceswhich have between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; or even more preferably, between about 91% andabout 99%; of amino acids which are identical or functionally equivalentto the amino acids of a piwi family protein or piwi family gene, will besequences which are “essentially the same”.

[0064] piwi family gene products and piwi family genes which havefunctionally equivalent codons are also covered by the invention. Theterm “functionally equivalent codon” is used herein to refer to codonsthat encode the same amino acid, such as the six codons for arginine orserine, and also to refer to codons that encode biologically equivalentamino acids (see Table 1). Thus, when referring to the sequence examplespresented in SEQ ID NOs:1-6, the substitution of functionally equivalentcodons of Table. 1 into the sequence examples of SEQ ID NOs:1-6 isenvisioned. Thus, applicants are in possession of amino acid and nucleicacids sequences which include such substitutions but which are not setforth herein in their entirety for convenience. TABLE I FunctionallyEquivalent Codons. Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGG UGU Aspartic Acid Asp D GAG GAU Glumatic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUG AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUG CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S ACG AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

[0065] It will also be understood that amino acid and nucleic acidsequences may include additional residues, such as additional N- orC-terminal amino acids or 5′ or 3′ sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences which may, for example, include variousnon-coding sequences flanking either of the 5′ or 3′ portions of thecoding region or may include various internal sequences, i.e., introns,which are known to occur within genes.

[0066] The present invention also encompasses the use of DNA segmentswhich are complementary, or essentially complementary, to the sequencesset forth in the specification. Nucleic acid sequences which are“complementary” are those which are base-pairing according to thestandard Watson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment in question under relativelystringent conditions such as those described herein. A particularexample of a contemplated complementary nucleic acid segment is anantisense oligonucleotide.

[0067] Nucleic acid hybridization will be affected by such conditions assalt concentration, temperature, or organic solvents, in addition to thebase composition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess of30° C., typically in excess of 37° C., and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1,000 mM,typically less than 500 mM, and preferably less than 200 mM. However,the combination of parameters is much more important than the measure ofany single parameter. (See, e.g., Wetmur & Davidson, 1968). Determiningappropriate hybridization conditions to identify and/or isolatesequences containing high levels of homology is well known in the art.(See e.g., Sambrook et al., 1989). For the purposes of specifyingconditions of high stringency, preferred conditions are saltconcentration of about 200 mM and temperature of about 45° C.

[0068] Probe sequences may also hybridize specifically to duplex DNAunder certain conditions to form triplex or other higher order DNAcomplexes. The preparation of such probes and suitable hybridizationconditions are well known in the art.

[0069] As used herein, the term “DNA segment” refers to a DNA moleculewhich has been isolated free of total genomic DNA of a particularspecies. Furthermore, a DNA segment encoding a piwi family polypeptiderefers to a DNA segment which contains piwi family coding sequences, yetis isolated away from, or purified free from, total genomic DNA of asource species, such as Homo sapiens. Included within the term “DNAsegment” are DNA segments and smaller fragments of such segments, andalso recombinant vectors, including, for example, plasmids, cosmids,phages, viruses, and the like.

[0070] Similarly, a DNA segment comprising an isolated or purified piwifamily gene refers to a DNA segment including piwi family codingsequences isolated substantially away from other naturally occurringgenes or protein encoding sequences. In this respect, the term “gene” isused for simplicity to refer to a functional protein, polypeptide orpeptide encoding unit. As will be understood by those in the art, thisfunctional term includes both genomic sequences and cDNA sequences.“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, the piwi family gene, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to the segment by the hand of man.

[0071] In particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a piwi family polypeptide that includes within its amino acidsequence an amino acid sequence selected from any of SEQ ID NOs:2, 4 and6. In other particular embodiments, the invention concerns isolated DNAsegments and recombinant vectors incorporating DNA sequences whichencode a protein that includes within its amino acid sequence the aminoacid sequence of a piwi family polypeptide corresponding to humantissues (referred to herein as HIWI).

[0072] It will also be understood that this invention is not limited tothe particular nucleic acid and amino acid sequences of any of SEQ IDNOs:1-6. Recombinant vectors and isolated DNA segments may thereforevariously include the piwi family polypeptide-encoding region itself,include coding regions bearing selected alterations or modifications inthe basic coding region, or include encoded larger polypeptides whichnevertheless include piwi family polypeptide-encoding regions or mayencode biologically functional equivalent proteins or peptides whichhave variant amino acid sequences.

[0073] In certain embodiments, the invention concerns isolated DNAsegments and recombinant vectors which encode a protein or peptide thatincludes within its amino acid sequence an amino acid sequenceessentially as set forth in any of SEQ ID NOs:2, 4 and 6. Naturally,where the DNA segment or vector encodes a full length piwi family geneproduct, the most preferred nucleic acid sequence is that which isessentially as set forth in any of SEQ ID NOs:1, 3 and 5 and whichencode a protein that exhibits activity in the modulation of growth,proliferation and self-renewing division of stem cells (in for examplegermline stem cells), and proliferation of primordial germ cells as maybe determined by, for example, immunofluorescence assays, as disclosedherein.

[0074] The term “a sequence essentially as set forth in any of SEQ IDNOs:2, 4 and 6″ means that the sequence substantially corresponds to aportion an amino acid sequence of any of SEQ ID NOs:2, 4 and 6 and hasrelatively few amino acids which are not identical to, or a biologicallyfunctional equivalent of, the amino acids of an amino acid sequence ofany of SEQ ID NOs:2, 4 and 6. The term “biologically functionalequivalent” is well understood in the art and is further defined indetail herein. Accordingly, sequences, which have between about 70% andabout 80%; or more preferably, between about 81% and about 90%; or evenmore preferably, between about 91% and about 99%; of amino acids whichare identical or functionally equivalent to the amino acids of any ofSEQ ID NOs:2, 4 and 6, will be sequences which “a sequence essentiallyas set forth in any of SEQ ID NOs:2, 4 and 6”.

[0075] In particular embodiments, the invention concerns gene therapymethods that use isolated DNA segments and recombinant vectorsincorporating DNA sequences which encode a protein that includes withinits amino-acid sequence an amino acid sequence of any of SEQ ID NOs:2, 4and 6, any of SEQ ID NOs:2, 4 and 6 including sequences which arederived from mammalian tissue, among other tissue. In other particularembodiments, the invention concerns isolated DNA sequences andrecombinant DNA vectors incorporating DNA sequences which encode aprotein that includes within its amino acid sequence the amino acidsequence of the piwi family protein from human tissue (HIWI).

[0076] In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in any of SEQ ID NOs:1, 3and 5. The term “a sequence essentially as set forth in any of SEQ IDNOs:1, 3 and 5” are used in the same sense as described above and meansthat the nucleic acid sequence substantially corresponds to a portion ofany of SEQ ID NOs:1, 3 and 5, respectively, and has relatively fewcodons which are not identical, or functionally equivalent, to thecodons of any of SEQ ID NOs:1, 3 and 5, respectively. Again, DNAsegments which encode gene products exhibiting activity in themodulation of growth, proliferation and self-renewing division of stemcells, and proliferation of primordial germ cells or other biologicalactivity of the piwi family gene product will be most preferred. Theterm “functionally equivalent codon” is used herein to refer to codonsthat encode the same amino acid, such as the six codons for arginine orserine, and also to refer to codons that encode biologically equivalentamino acids (see Table 1).

[0077] The nucleic acid segments of the present invention, regardless ofthe length of the coding sequence itself, may be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, nucleic acid fragments may beprepared which include a short stretch complementary to a nucleic acidsequence set for in any of SEQ ID NOs:1, 3 and 5 respectively, such asabout 10 nucleotides, and which are up to 10,000 or 5,000 base pairs inlength, with segments of 3,000 being preferred in certain cases. DNAsegments with total lengths of about 1,000, 500, 200, 100 and about 50base pairs in length are also contemplated to be useful.

[0078] The DNA segments of the present invention encompass biologicallyfunctional equivalent piwi family proteins and peptides. Such sequencesmay rise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded.

[0079] Alternatively, functionally equivalent proteins or peptides maybe created via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the, properties of the amino acids being exchanged,e.g. substitution of Ile for Leu at amino acid 13, at amino acid 106and/or at amino acid 785 for PIWI in SEQ ID NOs:1-2; substitution of Ilefor Leu at amino acid 90, at amino acid 383 and/or at amino acid 816 forMIWI in SEQ ID NOs:3-4; and substitution of lie for Leu at amino acid76, at amino acid 303 and/or at amino acid 735 for HIWI in SEQ IDNOs:5-6. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test piwi familymutants in order to examine activity in the modulation of growth,proliferation and self-renewing division of stem cells, andproliferation of primordial germ cells, or other activity at themolecular level.

[0080] If desired, one may also prepare fusion proteins and peptides,e.g., where the piwi family coding region is aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

[0081] Recombinant vectors form important further aspects of the presentinvention. Particularly useful vectors are contemplated to be thosevectors in which the coding portion of the DNA segment is positionedunder the control of a promoter. The promoter may be in the form of thepromoter which is naturally associated with the piwi family gene, e.g.,in mammalian tissues, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding segment or exon, for example,using recombinant cloning and/or PCR technology, in connection with thecompositions disclosed herein.

[0082] In other embodiments, it is contemplated that certain advantageswill be gained by positioning the coding. DNA segment under the controlof a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a piwi family gene in its naturalenvironment. Such promoters may include promoters isolated frombacterial, viral, eukaryotic, fish, avian or mammalian cells. Naturally,it will be important to employ a promoter that effectively directs theexpression of the DNA segment in the cell type chosen for expression.The use of promoter and cell type combinations for protein expression isgenerally known to those of skill in the art of molecular biology, forexample, see Sambrook et al., 1989, specifically incorporated herein byreference. The promoters employed may be constitutive, or inducible, andcan be used under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins or peptides. Appropriatepromoter systems contemplated for use in high-level expression include,but are not limited to, the vaccina virus promoter and the baculoviruspromoter.

[0083] In an alternative embodiment, the present invention provides anexpression vector comprising a polynucleotide that encodes a piwi familypolypeptide having activity in the modulation of growth, proliferationand self-renewing division of stem cells (including germline and somaticstem cells), cell proliferation (including activity in embryonicdevelopment as described in Example 1), proliferation of primordial germcells, or other biological activity in accordance with the presentinvention. Also preferably, an expression vector of the presentinvention comprises a polynucleotide that encodes human piwi family geneproduct (HIWI). More preferably, an expression vector of the presentinvention comprises a polynucleotide that encodes a polypeptidecomprising an amino acid residue sequence of any of SEQ ID NOs:2, 4 and6. More preferably, an expression vector of, the present inventioncomprises a polynucleotide comprising the nucleotide base sequence ofany of SEQ ID NOs:1, 3 and 5. Even more preferably, an expression vectorof the invention comprises a polynucleotide operatively linked to anenhancer-promoter. More preferably still, an expression vector of theinvention comprises a polynucleotide operatively linked to a prokaryoticpromoter. Alternatively, an expression vector of the present inventioncomprises a polynucleotide operatively linked to an enhancer-promoterthat is a eukaryotic promoter, and the expression vector furthercomprises a polyadenylation signal that is positioned 3′ of thecarboxy-terminal amino acid and within a transcriptional unit of theencoded polypeptide.

[0084] In yet another embodiment, the present invention provides arecombinant host cell transfected with a polynucleotide that encodes apiwi family polypeptide having activity in the modulation of growth,proliferation and self-renewing division of stem cells (includinggermline and somatic stem cells), cell proliferation (including activityin embryonic development as described in Example 1), proliferation ofprimordial germ cells, or other biological activity in accordance withthe present invention. SEQ ID NOs:3-6 set forth nucleotide and aminoacid sequences from representative vertebrates, human and mouse. Alsocontemplated by the present invention are homologous or biologicallyequivalent polynucleotides and piwi family polypeptides found in othervertebrates. Preferably, a recombinant host cell of the presentinvention is transfected with the polynucleotide that encodes human piwifamily polypeptide. More preferably, a recombinant host cell of thepresent invention is transfected with the polynucleotide sequence of SEQID NO:3 (miwi) or SEQ ID NO:5 (hiwi). Even more preferably, a host cellof the invention is a eukaryotic host cell. Still more preferably, arecombinant host cell of the present invention is a vertebrate cell.Preferably, a recombinant host cell of the invention is a mammalian,avian or fish cell.

[0085] In another-aspect, a recombinant host cell of the presentinvention is a prokaryotic host cell. Preferably, a recombinant hostcell of the invention is a bacterial cell, preferably a strain ofEscherichia coli. More preferably, a recombinant host cell comprises apolynucleotide under the transcriptional control of regulatory signalsfunctional in the recombinant host cell, wherein the regulatory signalsappropriately control expression of the piwi family polypeptide in amanner to enable all necessary transcriptional and post-transcriptionalmodification.

[0086] In yet another embodiment, the present invention contemplates amethod of preparing a piwi family polypeptide comprising transfecting acell with polynucleotide that encodes a piwi family polypeptide havingactivity in the modulation of growth, self-renewing division of stemcells (including germline and somatic stem cells), cell proliferation(including activity in embryonic development as described in theExamples), proliferation of primordial germ cells or other biologicalactivity in accordance with the present invention, to produce atransformed host cell; and maintaining the transformed host cell underbiological conditions sufficient for expression of the polypeptide. Morepreferably, the transformed host cell is a eukaryotic cell. Morepreferably still, the eukaryotic cell is a vertebrate cell.Alternatively, the host cell is a prokaryotic cell. More preferably, theprokaryotic cell is a bacterial cell of Escherichia coli. Even morepreferably, a polynucleotide transfected into the transformed cellcomprises the nucleotide base sequence of any of SEQ ID NOs:1, 3 and 5.SEQ ID NOs:3-6 set forth nucleotide and amino acid sequences forrepresentative vertebrates, mouse and human. Also contemplated by thepresent invention are homologues or biologically equivalent piwi familypolynucleotides and polypeptides found in other vertebrates,particularly warm blooded vertebrates.

[0087] As mentioned above, in connection with expression embodiments toprepare recombinant piwi family proteins and peptides, it iscontemplated that longer DNA segments will most often be used, with DNAsegments encoding the entire piwi family protein, functional domains orcleavage products thereof, being most preferred. However, it will beappreciated that the use of shorter DNA segments to direct theexpression of piwi family peptides or epitopic core regions, such as maybe used to generate anti-piwi family antibodies, also falls within thescope of the invention.

[0088] DNA segments which encode peptide antigens from about 15 to about50 amino acids in length, or more preferably, from about 15 to about 30amino acids in length are contemplated to be particularly useful. DNAsegments encoding peptides will generally have a minimum coding lengthin the order of about 45 to about 150, or to about 90 nucleotides. DNAsegments encoding full length proteins may have a minimum coding lengthon the order of about 4,000 nucleotides for a protein in accordance withany of SEQ ID NOs: 2,4 and 6.

[0089] Naturally, the present invention also encompasses DNA segmentswhich are complementary, or essentially complementary, to the sequencesset forth in any of SEQ ID NOs:1, 3 and 5. The terms “complementary” and“essentially complementary” are defined above. Excepting intronic orflanking regions, and allowing for the degeneracy of the genetic code,sequences which have between about 70% and about 80%; or morepreferably, between about 81% and about 90%; or even more preferably,between about 91% and about 99%; of nucleotides which are identical orfunctionally equivalent (i.e. encoding the same amino acid) ofnucleotides of any of SEQ ID NOs:1, 3 and 5, will be sequences which are“a sequence essentially as set forth in any of SEQ ID NOs:1, 3 and 5”.Sequences which are essentially the same as those set forth in any ofSEQ ID NOs:1, 3 and 5 may also be functionally defined as sequenceswhich are capable of hybridizing to a nucleic acid segment containingthe complement of any of SEQ ID NOs:1, 3 and 5 under relativelystringent conditions. Suitable relatively stringent hybridizationconditions are described herein and will be well known to those of skillin the art.

Biologically Functional Equivalents

[0090] As mentioned above, modification and changes may be made in thestructure of the piwi family proteins and peptides described herein andstill obtain a molecule having like or otherwise desirablecharacteristics. For example, certain amino acids may be substituted forother amino acids in a protein structure without appreciable loss ofinteractive capacity with structures such as, for example, in thenucleus of a cell. Since it is the interactive capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence (or, of course, its underlying DNA coding sequence) andnevertheless obtain a protein with like or even countervailingproperties (e.g., antagonistic v. agonistic). It is thus contemplated bythe inventor that various changes may be made in the sequence of thepiwi family proteins and peptides (or underlying DNA) withoutappreciable loss of their biological utility or activity.

[0091] It is also well understood by the skilled artisan that, inherentin the definition of a biologically functional equivalent protein orpeptide, is the concept that there is a limit to the number of changesthat may be made within a defined portion of the molecule and stillresult in a molecule with an acceptable level of equivalent biologicalactivity. Biologically functional equivalent peptides are thus definedherein as those peptides in which certain, not most or all, of the aminoacids may be substituted. Of course, a plurality of distinctproteins/peptides with different substitutions may easily be made andused in accordance with the invention.

[0092] It is also well understood that where certain residues are shownto be particularly important to the biological or structural propertiesof a protein or peptide, e.g., residues in active sites, such residuesmay not generally be exchanged. This is the case in the presentinvention, where if any changes, for example, in the PIWI box of piwifamily polypeptides, could result in a loss of an aspect of the utilityof the resulting peptide for the present invention.

[0093] Amino acid substitutions, such as those which might be employedin modifying the piwi family proteins and peptides described herein, aregenerally based on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. An analysis of the size, shape and type of the aminoacid side-chain substituents reveals that arginine, lysine and histidineare all positively charged residues; that alanine, glycine and serineare all a similar size; and that phenylalanine, tryptophan and tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and histidine; alanine, glycine andserine; and phenylalanine, tryptophan and tyrosine; are defined hereinas biologically functional equivalents.

[0094] In making such changes, the hydropathic index of amino acids maybe considered. Each amino acid has been assigned a hydropathic index onthe basis of their hydrophobicity and charge characteristics, these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

[0095] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 of the original value is preferred, those whichare within ±1 of the original value are particularly preferred, andthose within ±5 of the original value are even more particularlypreferred.

[0096] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

[0097] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan(−3.4).

[0098] In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ofthe original value is preferred, those which are within 1 of theoriginal value are particularly preferred, and those within ±0.5 of theoriginal value are even more particularly preferred.

[0099] While discussion has focused oh functionally equivalentpolypeptides arising from amino acid changes, it will be appreciatedthat these changes may be effected by alteration of the encoding DNA,taking into consideration also that the genetic code is degenerate andthat two or more codons may code for the same amino acid.

Sequence Modification Techniques

[0100] Modifications to the piwi family proteins and peptides describedherein may be carried out using techniques such as site directedmutagenesis. Site-specific mutagenesis is a technique useful in thepreparation of individual peptides, or biologically functionalequivalent proteins or peptides, through specific mutagenesis of theunderlying DNA. The technique further provides a ready ability toprepare and test sequence variants, for example, incorporating one ormore of the foregoing considerations, by introducing one or morenucleotide sequence changes into the DNA. Site-specific mutagenesisallows the production of mutants through the use of specificoligonucleotide sequences which encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 17 to 30 nucleotides in lengthis preferred, with about. 5 to 10 residues on both sides of the junctionof the sequence being altered.

[0101] In general, the technique of site-specific mutagenesis is wellknown in the art as exemplified by publications (e.g., Adelman et al.,1983). As will be appreciated, the technique typically employs a phagevector which exists in both a single stranded and double stranded form.Typical vectors useful in site-directed mutagenesis include vectors suchas the M13 phage (Messing et al., 1981). These phage are readilycommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis which eliminates the step of transferringthe gene of interest from a plasmid to a phage.

[0102] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes, for example, a human piwi familypolypeptide (HIWI). An oligonucleotide primer bearing the desiredmutated sequence is prepared, generally synthetically, for example bythe method of Crea et al. (1978). This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. Coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

[0103] The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful piwi family polypeptide or other species capable ofmodulating growth, proliferation and self-renewing division of stemcells, and proliferation of primordial germ cells and is not meant to belimiting as there are other ways in which sequence variants of thesepeptides may be obtained. For example, recombinant vectors encoding thedesired genes may be treated with mutagenic agents to obtain sequencevariants (see, e.g., a method described by Eichenlaub, 1979) for themutagenesis of plasmid DNA using hydroxylamine.

Other Structural Equivalents

[0104] In addition to the piwi family peptidyl compounds describedherein, the inventors also contemplate that other sterically similarcompounds may be formulated to mimic the key portions of the peptidestructure. Such compounds may be used in the same manner as the peptidesof the invention and hence are also functional equivalents. Thegeneration of a structural functional equivalent may be achieved by thetechniques of modeling and chemical design known to those of skill inthe art. It will be understood that all such sterically similarconstructs fall within the scope of the present invention.

[0105] Introduction of Gene Products

[0106] Where the gene itself is employed to introduce the gene products,a convenient method of introduction will be through the use of arecombinant vector which incorporates the desired gene, together withits associated control sequences. The preparation of recombinant vectorsis well known to those of skill in the art and described in manyreferences, such as, for example, Sambrook et al. (1989), specificallyincorporated herein by reference.

[0107] In vectors, it is' understood that the DNA coding sequences to beexpressed, in this case those encoding the piwi family gene products,are positioned adjacent to and under the control of a promoter. It isunderstood in the art that to bring a coding sequence under the controlof such a promoter, one generally positions the 5′ end of thetranscription initiation site of the transcriptional reading frame ofthe gene product to be expressed between about 1 and about 50nucleotides “downstream” of (i.e., 3′ of) the chosen promoter. One mayalso desire to incorporate into the transcriptional unit of the vectoran appropriate polyadenylation site (e.g., 5′-AATAAA-3′), if one was notcontained within the original inserted DNA. Typically, these poly Aaddition sites are placed about 30 to 2000 nucleotides “downstream” ofthe coding sequence at a position prior to transcription termination.

[0108] While use of the control sequences of the specific gene (i.e., apiwi family promoter for a piwi family gene) will be preferred, there isno reason why other control sequences could not be employed, so long asthey are compatible with the genotype of the cell being treated. Thus,one may mention other useful promoters by way of example, including,e.g., an SV40 early promoter, a long terminal repeat promoter fromretrovirus, an actin promoter, a heat shock promoter, a metallothioneinpromoter, and the like.

[0109] As is known in the art, a promoter is a region of a DNA moleculetypically within about 100 nucleotide pairs in front of (upstream of)the point at which transcription begins (i.e., a transcription startsite). That region typically contains several types of DNA sequenceelements that are located in similar relative positions in differentgenes. As used herein, the term “promoter” includes what is referred toin the art as an upstream promoter region, a promoter region or apromoter of a generalized eukaryotic RNA Polymerase II transcriptionunit.

[0110] Another type of discrete transcription regulatory sequenceelement is an enhancer. An enhancer provides specificity of time,location and expression level for a particular encoding region (e.g.,gene). A major function of an enhancer is to increase the level oftranscription of a coding sequence in a cell that contains one or moretranscription factors that bind to that enhancer. Unlike a promoter, anenhancer can function when located at variable distances fromtranscription start sites so long as a promoter is present.

[0111] As used herein, the phrase “enhancer-promoter” means a compositeunit that contains both enhancer and promoter elements. Anenhancer-promoter is operatively linked to a coding sequence thatencodes at least one gene product. As used herein, the phrase“operatively linked” means that an enhancer-promoter is connected to acoding sequence in such a way that the transcription of that codingsequence is controlled and regulated by that enhancer-promoter. Meansfor operatively linking an enhancer-promoter to a coding sequence arewell known in the art. As is also well known in the art, the preciseorientation and location relative to a coding sequence whosetranscription is controlled, is dependent inter alia upon the specificnature of the enhancer-promoter. Thus, a TATA box minimal promoter istypically located from about 25 to about 30 base pairs upstream of atranscription initiation site and an upstream promoter element istypically located from about 100 to about 200 base pairs upstream of atranscription initiation site. In contrast, an enhancer can be locateddownstream from the initiation site and can be at a considerabledistance from that site.

[0112] An enhancer-promoter used in a vector construct of the presentinvention can be any enhancer-promoter that drives expression in a cellto be transfected. By employing an enhancer-promoter with well-knownproperties, the level and pattern of gene product expression can beoptimized.

[0113] For introduction of, for example, the human piwi family gene(hiwi), it is proposed that one will desire to preferably employ avector construct that will deliver the desired gene to the affectedcells. This will, of course, generally require that the construct bedelivered to the targeted cells, for example, mammalian, avian or fishgermline stem cells. It is proposed that this may be achieved mostpreferably by introduction of the desired gene through the use of aviral vector to carry the piwi family sequence to efficiently infect thecells. These vectors will preferably be an adenoviral, a retroviral, avaccinia viral vector or adeno-associated virus. These vectors arepreferred because they have been successfully used to deliver desiredsequences to cells and tend to have a high infection efficiency.Suitable vector-piwi family gene constructs are adapted foradministration as pharmaceutical compositions, as described hereinbelow.

[0114] Commonly used viral promoters for expression vectors are derivedfrom polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40).The early and late promoters of SV40 virus are particularly usefulbecause both are obtained easily from the virus as a fragment which alsocontains the SV40 viral origin of replication. Smaller or larger SV40fragments may also be used, provided there is included the approximately250 bp sequence extending from the Hind III site toward the Bgl I sitelocated in the viral origin of replication.

[0115] Further, it is also possible, and often desirable, to utilizepromoter or control sequences normally associated with the desired genesequence, provided such control sequences are compatible with the hostcell systems.

[0116] The origin of replication may be provided either by constructionof the vector to include an exogenous origin, such as may be derivedfrom SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or maybe provided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter is oftensufficient.

[0117] Where a piwi family gene itself is employed it will be mostconvenient to simply use a wild type piwi family gene directly. However,it is contemplated that certain regions of a piwi family gene may beemployed exclusively without employing an entire wild type piwi familygene. It is proposed that it will ultimately be preferable to employ thesmallest region needed to modulate stem cell growth, proliferation andself-renewing division, and/or primordial germ cell proliferation, sothat one is not introducing unnecessary DNA into cells which receive apiwi family gene construct. Techniques well known to those of skill inthe art, such as the use of restriction enzymes, will allow for thegeneration of small regions of an exemplary piwi family gene. Theability of these regions to modulate cell signaling can easily bedetermined by the assays reported in the Examples. In general,techniques for assessing the modulation of stem cell growth,proliferation and self-renewing division, along with primordial germcell proliferation are known in the art.

Cell Culture Techniques

[0118] As disclosed herein, PIWI functions both as a cytoplasmic protein(during embryonic germline development) and as nuclear protein duringstem cell division. PIWI has a cell-autonomous function to promote stemcell division in addition to its signaling function in maintaining stemcells. The dose of PIWI gene determines the number of germline stemcells formed in the embryo.

[0119] In accordance with the present invention, piwi family genes andgene products can thus be used to expand a primitive cell population invitro. The term “primitive cell” is adopted herein for convenience tocollectively refer to stem cells, primordial germ cells and to otherprimitive cells having totipotent or pluripotent characteristics, orother characteristics of stem cells and primordial germ cells. The term“stem cell” is meant to have its art-recognized meaning, and to include,but not be limited to, the following examples of stem cells:hematopoietic stem cells, neural stem cells, and germline stem cells.The term “primordial germ cell” is also meant to have its art-recognizedmeaning. Such expanded primitive cell populations are utilized fortherapeutic or genetic engineering purposes in human and mammalian,avian, and fish systems.

[0120] The term “sustained” as used herein with respect to primitivecells, and cultures of the same, refers to a cell or cell culturecapable of undergoing further cell division, even if the cells areeventually subject to senescence.

[0121] A method of culturing primitive cells is thus envisioned inaccordance with the present invention. The method comprises providing aculture comprising a primitive cell, and delivering to the provided cella piwi family gene product, whereby growth of the cell in the culture isaccomplished. Preferably, the primitive cell is maintained in culturefor a time sufficient to produce a sustained cell culture.

[0122] Optionally, the piwi family gene product is delivered to theprimitive cell by delivering to the primitive cell an effective amountof a DNA molecule comprising a polynucleotide that encodes abiologically active piwi family polypeptide; and maintaining theprimitive cell under conditions sufficient for expression of thepolypeptide. Alternatively, the piwi family gene product is delivered tothe cell by administration of a biologically active piwi familypolypeptide to the culture via the culture media or via a feeder matrixincluded in the culture.

[0123] A preferred embodiment of the method comprises: (a) collectingprimitive cells; (b) depositing the primitive cells in contact with afeeder matrix, the feeder matrix comprising a piwi family gene product;and (c) growing the primitive cells on the feeder matrix in the presenceof media for a time sufficient to produce a sustained primitive cellculture.

[0124] Thus, typically, the cultures of the present invention include afeeder matrix as defined herein. A feeder matrix can either be cells orcell lines cultured for the purpose of obtaining a sustained primitivecell culture. In this case the cell line can be transformed with a piwifamily gene product in accordance with techniques disclosed herein so asto produce the piwi family gene product. Alternatively, a feeder matrixcan be derived from or provided by the organ or tissue in which theprimitive cells are located, e.g., the gonad. Alternatively, the feedercells comprising the feeder matrix could be substituted withextracellular matrix plus bound growth factors, and particularlyincluding a piwi family gene product.

[0125] A feeder matrix as used herein is constructed in accordance withprocedures known in the art. As noted above, it is preferred that thefeeder matrix be preconditioned. By the term “preconditioned” it ismeant that the feeder matrix is cultured in the presence of media for aperiod of time prior to the depositing of primitive cells in contactwith the feeder matrix, e.g. a time sufficient to initiate and establishproduction of, for example, growth factors or other factors by thefeeder matrix, and particularly including a piwi family gene product.Thus, a feeder matrix can be preconditioned by culturing the feedermatrix by itself for one to two days prior to the depositing ofprimitive cells in contact with the feeder matrix.

[0126] Candidate cells for use in the feeder matrix are disclosed in theExamples. Additionally, the feeder matrix can comprise Sertoli cells orfibroblast cells, including mouse fibroblast cells. STO fibroblasts andprimary fibroblasts are suitable examples. Also, it is contemplated thatfeeder matrices comprising cells from other murine species (e.g., rat);other mammalian species (e.g., ungulate, bovine, and porcine species);or avian species (e.g. Gallinacea, chicken, turkey, duck, goose, quailand pheasant) may also be used.

[0127] The culturing method of the present invention can optionallycomprise establishing a monolayer of feeder cells. Feeder cells maymitotically inactivated using standard techniques. For example, thefeeder cells may be exposed to gamma radiation (e.g. 4000 Rads of gammaradiation) or may be treated with Mitomycin C (e.g. 10 μg/ml for 2-3hours). Procedures for mitotically inactivating cells are also detailedin the information typically sent with cells from the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.2011 Q-2209 (e.g. STO feeder cells are available under ATCC accessionnumber 1503). Monolayers may optionally be cultured to about 80%confluency, preferably to about 90% confluency, and more preferablyabout 100% confluency. While configuration of the feeder cells as amonolayer is the preferred configuration for the culture, any suitableconfiguration is contemplated to be within the scope of the presentinvention.

[0128] Thus, for example, layers, monolayers, clusters, aggregates orother associations or groupings of feeder cells are contemplated to fallwithin the scope of the present invention and are particularlycontemplated to fall with the meaning of the term “matrix”.

[0129] Thus, the primitive cells can extend non-uniformly through thefeeder cells vertically, horizontally, diagonally, or in any combinationthereof, such that cell aggregates are formed. In accordance with thepresent invention, it is thus contemplated that the cultures can beformed by mixing primitive cells with appropriate feeder cells andinserting the mixture into a suitable culture container, such as amulti-well plate.

[0130] The media used in carrying out the present invention may be anysuitable media. The media may be a conditioned media or a syntheticmedia, both of which are known in the art. Conditioned media, andparticularly BRL conditioned media, is currently preferred. By way ofexample, BRL conditioned media is prepared according to art-recognizedtechniques, such as described by Smith, A. G. and Hooper, M. L., Dev.Biol. 1987 May; 121(1):1-9. BRL cells are available from ATCC underaccession number CRL-1442. The media can be supplemented with a piwifamily gene product. The media can also be supplemented with growthfactors, including but not limited to leukemia inhibitory factor (LIF),insulin-like growth factor (IGF), fibroblast growth factor (FGF), basicfibroblast growth factor (bFGF), stem cell factor (SCF—also called steelfactor or SF), transforming growth factor-β1 (TGF-β1) and anti-retinoicacid.

[0131] Cell cultures of the present invention may be formulated foradministration to animals by dissociating the cells (e.g., by mechanicaldissociation) and intimately admixing the cells with a pharmaceuticallyacceptable carrier (e.g., phosphate-buffered saline (PBS) solution).Primitive cells in such formulations may be prepared to carry aheterologous DNA sequence into a subject, preferably a vertebratesubject and more preferably a warm-blooded vertebrate subject, in themanner described in greater detail below.

[0132] The primitive cells cultured in accordance with the presentinvention are useful, among other things, as a tool for the study ofembryological development (i.e., by labeling the cells with a markergene and observing their distribution after injection in vivo) and theproduction of transgenic animals. They are useful in allowing theapplication of homologous recombination to the production of transgenicanimals.

[0133] The primitive cells should also be useful for the production ofchimeric animals, including transgenic chimeric animals. It iscontemplated that chimeric or transgenic chimeric non-human animals,including particularly chimeric or transgenic chimeric avians (i.e.birds), will be useful for the recovery of heterologous proteins fromthe animals. In the case of an avian, the protein can be recovereddirectly from the eggs of such chimeric or transgenic chimeric avians.Such avians can be thus be used for the production and recovery oftherapeutic proteins and other polypeptides, including piwi familypolypeptides themselves.

[0134] The following U.S. Patents pertain to the culturing of stem cellsan are used herein to illuminate the background of the invention, and inparticular cases, to provide additional details respecting the practice.Each of the following U.S. Patent is incorporated herein by reference inits entirety: U.S. Pat. No. 5,690,926 issued Nov. 25, 1997 to Hogan;U.S. Pat. No. 5,670,372 issued Sep. 23, 1997 to Hogan; U.S. Pat. No.5,537,357 issued Sep. 26, 1995 to Hogan; U.S. Pat. No. 5,340,740 issuedAug. 23, 1994 to Petitte et al.; U.S. Pat. No. 5,656,479 issued Aug. 12,1997 to Petitte et al.; and U.S. Pat. No. 5,830,510 issued Nov. 3, 1998to Petitte et al.

Transgenic Animals

[0135] It is also contemplated to be within the scope of the presentinvention to prepare a transgenic non-human animal which expresses apiwi family gene of the present invention. A preferred transgenic animalis a mouse.

[0136] The term “transgene” refers to exogenous genetic material whichdoes not naturally form part of the genetic material of an animal to begenetically altered but can be incorporated into the germ and/or somaticcells of that animal by standard transgenic techniques. The term“heterologous DNA” refers to DNA which has been transferred from oneindividual animal, species or breed to a different individual animal,species or breed. The term “transgenic” refers to cells, tissues,embryos, fetuses or animals which carry one or more transgenes. The term“chimeric” refers to an embryo, fetus or animal which consists of two ormore tissues of different genetic composition.

[0137] Techniques for the preparation of transgenic animals are known inthe art. Exemplary techniques are described in U.S. Pat. No. 5,489,742(transgenic rats); U.S. Pat. Nos. 4,736,866, 5,550,316, 5,614,396,5,625,125, and 5,648,061 (transgenic mice); U.S. Pat. No. 5,573,933(transgenic pigs); 5,162,215 (transgenic avian species) and U.S. Pat.No. 5,741,957 (transgenic bovine species), the entire contents of eachof which are herein incorporated by reference.

[0138] With respect to a representative method for the preparation of atransgenic mouse, cloned recombinant or synthetic DNA sequences or DNAsegments encoding a piwi family gene product are injected intofertilized mouse eggs (e.g. an embryo). The injected eggs are implantedin pseudo pregnant females and are grown to term to provide transgenicmice whose cells express a piwi family gene product. Preferably, theinjected sequences are constructed having promoter sequences connectedso as to express the desired protein in germline stem cells or otherprimitive cells of the transgenic mouse.

[0139] Fish represent a category of animals of interest for agriculturaland ecological reasons. Representative fish species include, but are notlimited to, trout, salmon, carp, shark, ray, flounder, sole, tilapia,medaka, goldfish, guppy, molly, platyfish, swordtail, zebrafish, loach,catfish, and the like. Representative methods for the preparation of atransgenic fish have been described by Ozato et al, Cell Differ.,19:237-244 (1986), Inoue et al, Cell Differ. Dev., 29:123-128 (1990),Rokkones et al, J. Comp. Physiol. B, 158:751-758 (1989), and Guyomard etal, Biochimie, 71:857-863 (1989), describing preparation of transgenicmedaka, medaka, salmon and trout, respectively.

[0140] The term “avian” as used herein refers to any avian species,including but not limited to Gallinacea sp., chicken, turkey, duck,goose, quail and pheasant. Chicken is currently preferred.

[0141] With respect to an exemplary method for the preparation of atransgenic avian species, certain donor cell types have been isolatedthat retain viability when injected into recipient embryos. See Etcheset al., Poultry Science 72:882-887 (1993); Etches et al., in AvianIncubation, Chapter 22, Butterworth Publishers (1990); Verrinder Gibbinset al., Fourth World Congress on Genetics Applied to LivestockProduction, Edinburgh, (1990); Petitte et al., Development 108, 185-189(1990)). These studies showed that blastodermal cells derived from StageX embryos (embryo at oviposition) remained viable when transferred tocomparable recipient Stage X embryos.

[0142] Thus, the present invention provides a new method of altering thephenotype of an animal, such as a bird, and the animals-so produced withthe primitive cells cultured in accordance with techniques disclosedherein. The method comprises transfecting primitive cells cultured inaccordance with the methods disclosed herein with the DNA sequence invitro (e.g., by electroporation or transformation with a retroviralvector), and then injecting the transfected primitive cells into anembryo, (e.g. an egg containing an embryonic bird via the yolk sac oronto the chorioallantoic membrane), with the DNA sequence beingeffective to cause a change in phenotype in the embryonic animal (e.g.,a change in protein expression, a change in growth rate, feedefficiency, disease resistance, or a combination of all of thesefactors). Preferably, the primitive cells are also transfected with apiwi family gene to facilitate production of germ cells in thetransgenic or chimeric bird and thereby also facilitate germlinetransmission of the DNA sequence of interest.

[0143] Preferably, in the case of a bird, the egg into which the DNA isintroduced is incubated to hatch, and the bird so produced is raised toat least an age at which the change in phenotype is expressed. It is ofno deleterious consequence if the transformed embryo and bird ischimeric, so long as a physiological response is achieved in the animalafter hatch sufficient to evoke a measurable phenotypic change measuredin any suitable way. By way of additional example, protein expression inthe chimera, or transgenic offspring thereof produced by germ linetransmission of the DNA sequence, may be established throughincorporation of an inducible promoter into the introduced DNA sequencecoupled with treatment of the chimera, or transgenic offspring thereof,with an agent which induces the promoter.

[0144] The mechanism of in ovo injection is not critical, but it ispreferred that the method not unduly damage the tissues and organs ofthe embryo or the extraembryonic membranes surrounding it so that thetreatment will not decrease hatch rate. For example, in ovo injectionscan be carried out by forming a window in a bird egg and then using amicro-pipette or needle for the injection, such a 50 μm-diametermicro-pipette or a 50 μm-diameter needle. If desired, the bird egg canbe sealed with a substantially bacteria-impermeable sealing materialsuch as wax or the like to prevent subsequent entry of undesirablebacteria. Suitable sealing material may optionally comprise biologicallyacceptable antimicrobial compositions.

[0145] The DNA sequence introduced with the primitive cells inaccordance with the present invention is, in general, a constructcomprised of a promoter functional in the cells of the candidate animaland a gene encoding a peptide or protein operably linked to thepromoter. Preferably, the protein or peptide is physiologically activeand capable of producing a phenotypic change in the animal. In general,the DNA construct may be a linear DNA sequence (introduced into theprimitive cells of the invention by electroporation) or a sequencecarried by a vector or other suitable carrier for transforming theprimitive cells of the invention using various techniques known in theart, such as liposomes, calcium phosphate, electroporation, or DMSO.Vectors, as discussed herein, may be plasmids, viruses (includingretroviruses), and phage, whether in native form or derivatives thereof.

[0146] The production of cloned genes, recombinant DNA, vectors,transformed host cells, proteins and protein fragments by geneticengineering is well known. See e.g., U.S. Pat. No. 4,761,371 to Bell etal. at Col. 6 line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clarket al. at Col. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 toSchilling at Col. 3 line 26 to Col. 14 line 12. Protocols forrestriction endonuclease digestion, preparation of vectors, DNApurification and other such procedures are essentially as described instandard cloning manuals. See Sambrook et al., Molecular Cloning, aLaboratory Manual, (2d Ed., Cold Spring Harbor Press, N.Y. (1989)).

[0147] As disclosed herein, a vector is a replicable DNA construct usedherein to either amplify and/or express DNA encoding the gene ofinterest. A suitable expression vector will have controlling elementscapable of expressing the cloned cDNA or genomic DNA placed in thecorrect orientation when the vector is introduced into the correct host.Such elements typically include but are not limited to a promoter regionwhich interacts specifically with cellular proteins involved intranscription or which may be inducible by administration of an inducingagent, enhancer elements which can stimulate transcription many-foldfrom linked heterologous promoters, a splice acceptor and/or donorsequence, and termination and polyadenylation signals. Also required isthe sequence for a ribosome binding site capable of permittingtranslation and which is operably linked to the gene to be expressed.Recently, a muscle-specific promoter has been isolated which ispositioned upstream of both the skeletal muscle structural gene and theessential proximal promoter element and is operably associated witheach. (Mar and Ordahl, Proc. Natl. Acad. Sci. USA 85, 6404-6408 (1988)).Vectors comprise plasmids, viruses (e.g. adenovirus, cytomegalovirus),phage, and DNA fragments integratable into the host genome byrecombination. The vector may optionally replicate and functionindependently of the host genome, or may in some instances integrateinto the genome itself.

[0148] The piwi family genes of the present invention can thus be usedto create transgenic animals with increased number of primordial germcells. Such animals in mammalian, avian, and fish systems should providean improved platform for germline-mediated gene transfer to createtransgenic stocks.

[0149] The genetically modified animals could express transgenes ofcommercial interest, including those having therapeutic or prophylacticvalue to the animal itself or to its offspring. For example, thetransgene of interest can encode a protein or peptide including, but notlimited to, growth hormone, thyroid releasing hormone (TRH), Marek'sMDX, and immunogenic recombinant antigens such as that for coccidiosis.

[0150] Alternatively, the genetically modified animals can be employedin the production of therapeutic agents. For example, geneticallymodified avian species that can lay eggs containing drugs, proteins andantibodies to ward off illness that have been produced via the insertionthe genes that make the proteins into viral and other vectors to getthem into the birds piwi family gene products can also be produced andisolated in this manner.

[0151] In accordance with, the present invention, piwi family genes canalso be inserted into such genetically modified animals to createanimals with increased number of primordial germ cells, therebyenhancing the likelihood of germline transmission of the transgeneencoding the therapeutic agent due to the increased number of primordialgerm cells.

Pharmaceutical Compositions

[0152] In a preferred embodiment, the present invention providespharmaceutical compositions comprising a polypeptide or polynucleotideof the present invention and a physiologically acceptable carrier. Morepreferably, a pharmaceutical composition comprises a piwi familypolypeptide or a polynucleotide that encodes those polypeptides.

[0153] A composition of the present invention is typically administeredparenterally in dosage unit formulations containing standard, well-knownnontoxic physiologically acceptable carriers, adjuvants, and vehicles asdesired. The term “parenteral” as used herein includes intravenous,intramuscular, intra-arterial injection, or infusion techniques.

[0154] Injectable preparations, for example sterile injectable aqueousor oleaginous suspensions, are formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol.

[0155] Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. In addition, fattyacids such as oleic acid find use in the preparation of injectables.

[0156] Preferred carriers include neutral saline solutions buffered withphosphate, lactate, Tris, and the like. Of course, one purifies thevector sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering adenovirus particles orendotoxins and other pyrogens such that it does not cause any untowardreactions in the individual receiving the vector construct. A preferredmeans of purifying the vector involves the use of buoyant densitygradients, such as cesium chloride gradient centrifugation.

[0157] A transfected cell can also serve as a carrier. By way ofexample, a liver cell can be removed from an organism, transfected witha polynucleotide of the present invention using methods set forth aboveand then the transfected cell returned to the organism (e.g. injectedintra-vascularly).

Generation of Antibodies

[0158] In still another embodiment, the present invention provides anantibody immunoreactive with a polypeptide of the present invention.Preferably, an antibody of the invention is a monoclonal antibody. Meansfor preparing and characterizing antibodies are well known in the art(See, e.g., Antibodies A Laboratory Manual, E. Howell and D. Lane, ColdSpring Harbor Laboratory, 1988).

[0159] Briefly, a polyclonal antibody is prepared by immunizing ananimal with an immunogen comprising a polypeptide or polynucleotide ofthe present invention, and collecting antisera from that immunizedanimal. A wide range of animal species can be used for the production ofantisera. Typically an animal used for production of anti-antisera is arabbit, a mouse, a rat, a hamster or a guinea pig. Because of therelatively large blood volume of rabbits, a rabbit is a preferred choicefor production of polyclonal antibodies.

[0160] As is well known in the art, a given polypeptide orpolynucleotide may vary in its immunogenicity. It is often necessarytherefore to couple the immunogen (e.g., a polypeptide orpolynucleotide) of the present invention) with a carrier. Exemplary andpreferred carriers are keyhole limpet hemocyanin (KLH) and bovine serumalbumin (BSA). Other albumins such as ovalbumin, mouse serum albumin orrabbit serum albumin can also be used as carriers.

[0161] Means for conjugating a polypeptide or a polynucleotide to acarrier protein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

[0162] As is also well known in the art, immunogenicity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete Freund's adjuvant, incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

[0163] The amount of immunogen used of the production of polyclonalantibodies varies, inter alia, upon the nature of the immunogen as wellas the animal used for immunization. A variety of routes can be used toadminister the immunogen, e.g. subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal. The production of polyclonal antibodiesis monitored by sampling blood of the immunized animal at various pointsfollowing immunization. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored.

[0164] In another aspect, the present invention contemplates a method ofproducing an antibody immunoreactive with a piwi family polypeptide, themethod comprising the steps of (a) transfecting recombinant host cellswith a polynucleotide that encodes that polypeptide; (b) culturing thehost cells under conditions sufficient for expression of thepolypeptide; (c) recovering the polypeptide; and (d) preparingantibodies to the polypeptide. Preferably, the piwi family polypeptidepossess a biological activity in accordance with the present invention.Even more preferably, the present invention provides antibodies preparedaccording to the method described above.

[0165] A monoclonal antibody of the present invention can be readilyprepared through use of well-known techniques such as those exemplifiedin U.S. Pat. No. 4,196,265, herein incorporated by reference. Typically,a technique involves first immunizing a suitable animal with a selectedantigen (e.g., a polypeptide or polynucleotide of the present invention)in a manner sufficient to provide an immune response. Rodents such asmice and rats are preferred animals. Spleen cells from the immunizedanimal are then fused with cells of an immortal myeloma cell. Where theimmunized animal is a mouse, a preferred myeloma cell is a murine NS-1myeloma cell.

[0166] The fused spleen/myeloma cells are cultured in a selective mediumto select fused spleen/myeloma cells from the parental cells. Fusedcells are separated from the mixture of non-fused parental cells, forexample, by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the media is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, the mediais supplemented with hypoxanthine.

[0167] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants forreactivity with an antigen-polypeptides. The selected clones can then bepropagated indefinitely to provide the monoclonal antibody.

[0168] By way of specific example, to produce an antibody of the presentinvention, mice are injected intraperitoneally with between about 1-200μg of an antigen comprising a polypeptide of the present invention. Blymphocyte cells are stimulated to grow by injecting the antigen inassociation with an adjuvant such as complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis). At some time (e.g., at least two weeks)after the first injection, mice are boosted by injection with a seconddose of the antigen mixed with incomplete Freund's adjuvant.

[0169] A few weeks after the second injection, mice are tail bled andthe sera, titered by immunoprecipitation against radiolabeled antigen.Preferably, the method of boosting and titering is repeated until asuitable titer is achieved. The spleen of the mouse with the highesttiter is removed and the spleen lymphocytes are obtained by homogenizingthe spleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0170] Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

[0171] Myeloma cells are combined under conditions appropriate to fosterfusion with the normal antibody-producing cells from the spleen of themouse or rat injected with the antigen/polypeptide of the presentinvention. Fusion conditions include, for example, the presence ofpolyethylene glycol. The resulting fused cells are hybridoma cells. Likemyeloma cells, hybridoma cells grow indefinitely in culture.

[0172] Hybridoma cells are separated from unfused myeloma cells byculturing in a selection medium such as HAT media (hypoxanthine,aminopterin, thymidine). Unfused myeloma cells lack the enzymesnecessary to synthesize nucleotides from the salvage pathway becausethey are killed in the presence of aminopterin, methotrexate, orazaserine. Unfused lymphocytes also do not continue to grow in tissueculture. Thus, only cells that have successfully fused (hybridoma cells)can grow in the selection media.

[0173] Each of the surviving hybridoma cells produces a single antibody.These cells are then screened for the production of the specificantibody immunoreactive with an antigen/polypeptide of the presentinvention. Single cell hybridomas are isolated by limiting dilutions ofthe hybridomas. The hybridomas are serially diluted many times and,after the dilutions are allowed to grow, the supernatant is tested forthe presence of the monoclonal antibody. The clones producing thatantibody are then cultured in large amounts to produce an antibody ofthe present invention in convenient quantity.

[0174] By use of a monoclonal antibody of the present invention,specific polypeptides and polynucleotide of the invention can berecognized as antigens, and thus identified. Once identified, thosepolypeptides and polynucleotide can be isolated and purified bytechniques such as antibody-affinity chromatography. Inantibody-affinity chromatography, a monoclonal antibody is bound to asolid substrate and exposed to a solution containing the desiredantigen. The antigen is removed from the solution through animmunospecific reaction with the bound antibody. The polypeptide orpolynucleotide is then easily removed from the substrate and purified.

Screening Assays

[0175] In yet another aspect, the present invention contemplates amethod of screening substances for their ability to affect or modulatethe biological activity of piwi family gene products. The presentinvention also contemplates a method of screening substances for theirability to affect or modulate the biological activity of piwi familygene products to thereby affect or modulate the growth, proliferationand self-renewing division, of stem cells, particularly germline stemcells, and proliferation of primordial germ cells. Utilizing the methodsand compositions of the present invention, screening assays for thetesting of candidate substances can be derived. A candidate substance isa substance which potentially can promote or inhibit the biologicalactivity of a piwi family gene product by binding, or otherintermolecular interaction, with the piwi family gene product.

[0176] An exemplary method of screening candidate substances for theirability to modulate piwi family biological activity comprises the stepsof: (a) establishing replicate test and control samples that comprise abiologically active piwi family polypeptide; (b) administering acandidate substance to test sample but not the control sample; (c)measuring the biological activity of the piwi family polypeptide in thetest and the control samples; and (d) determining that the candidatesubstance modulates piwi family biological activity if the biologicalactivity of the piwi family polypeptide measured for the test sample isgreater or less than the biological activity of the piwi familypolypeptide measured for the control sample. The biological activitiesof piwi family that may optionally be examined in connection with ascreening assay of the present invention comprise modulating the growth,proliferation and self-renewing division of stem cells, particularlygermline stem cells, proliferation of primordial germ cells or otherbiological activity in accordance with the present invention.

[0177] The replicate test and control samples can further comprise acell that expresses a biologically active piwi family polypeptide. Thepresent invention also contemplates a recombinant cell line suitable foruse in the exemplary method. A candidate substance identified accordingto the screening assay described herein is contemplated to have theability to modulate piwi family biological activity. Such as candidatecompound has utility in the treatment of disorders and conditionsassociated with the biological activity of piwi family.

[0178] In a cell-free system, the method comprises the steps ofestablishing a control system comprising a piwi family polypeptide and aligand wherein the piwi family polypeptide is capable of binding to theligand; establishing a test system comprising the piwi familypolypeptide, the ligand, and a candidate compound; measuring the bindingaffinity of the piwi family polypeptide and the ligand in the controland the test systems; and determining that the candidate compoundmodulates piwi family activity in a cell-free system if the bindingaffinity measured for the test system is less than or greater than thebinding affinity measured for the control system. An exemplary ligandcomprises a monoclonal antibody.

[0179] A screening assay of the present invention may also involvedetermining the ability of a candidate substance to modulate, i.e.inhibit or promote piwi family biological activity and preferably, tothereby modulate the growth, proliferation and self-renewing division ofstem cells, particularly germline stem cells, proliferation ofprimordial germ cells or division of other target cells. Target cellscan be either naturally occurring cells known to contain a polypeptideof the present invention or transformed cell produced in accordance witha method of transformation set forth hereinbefore.

[0180] As is well known in the art, a screening assay provides a cellunder conditions suitable for testing the modulation of piwi familybiological activity or of growth, proliferation and self-renewingdivision of stem cells, particularly germline stem cells, or ofproliferation of primordial germ cells. These conditions include but arenot limited to pH, temperature, tonicity, the presence of relevantmetabolic factors (e.g., metal ions such as for example Ca⁺⁺, growthfactor, interleukins, or colony stimulating factors), and relevantmodifications to the polypeptide such as glycosylation or prenylation.It is contemplated that a polypeptide of the present invention can beexpressed and utilized in a prokaryotic or eukaryotic cell. The hostcell can also be fractionated into sub-cellular fractions where thereceptor can be found. For example, cells expressing the polypeptide canbe fractionated into the nuclei, the endoplasmic reticulum, vesicles, orthe membrane surfaces of the cell.

[0181] In a preferred embodiment, temperature is from about 20° C. toabout 50° C., more preferably from about 30° C. to about 40° C. and,even more preferably about 37° C. Osmolality is preferably from about 5milliosmols per liter (mosm/L) to about 400 mosm/L and, more preferablyfrom about 200 milliosmols per liter to about 400 mosm/L and, even morepreferably from about 290 mosm/L to about 310 mosm/L. The presence offactors can be required for the proper testing of piwi family biologicalactivity modulation in specific cells. Such factors include, forexample, the presence and absence (withdrawal) of growth factor,interleukins, or colony stimulating factors. U.S. Pat. Nos. 5,837,479;5,645,999; 5,786,152; 5,739,278; and 5,352,660 also describe exemplaryscreening assays, and the entire contents of each are hereinincorporated by reference.

[0182] In one embodiment, a screening assay is designed to be capable ofdiscriminating candidate substances having selective ability to interactwith one or more of the polypeptides of the present invention but whichpolypeptides are without a substantially overlapping activity withanother of those polypeptides identified herein. Exemplary assaysincluding genetic screening assays and molecular biology screens such asa yeast two-hybrid screen which will effectively identifypiwi-interacting genes important for piwi-mediated cell growth,proliferation and self-renewing division processes. One version of theyeast two-hybrid-system has been described (Chien et al., 1991, Proc.Nail. Acad. Sci. USA, 88:9578-9582) and is commercially available fromClontech, Palo Alto, Calif.

Therapeutic Methods

[0183] As used herein, the terms “piwi family activity” and “piwi familybiological activity” are meant to be synonymous and are meant to referto any biological activity of a piwi family polypeptide. Exemplarybiological activities of piwi family comprise activity in the modulationof growth, proliferation and self-renewing division of stem cells(including germline and somatic stern cells), cell proliferation(including activity in embryonic development as described in theExamples), proliferation of primordial germ cells or other biologicalactivity in accordance with the present invention. The biologicalactivity can be accomplished by endogenous piwi family polypeptides orby piwi family polypeptide administered to a subject. Indeed, anisolated and purified piwi family polypeptide, recombinant piwi familypolypeptide, and/or piwi family analog or peptidomimetic, each preparedas described herein, can administered to a subject to impart piwi familybiological activity in the subject and to treat a disorder associatedwith piwi family biological activity in the subject. In such case theimparted piwi family biological activity comprises a piwi familybiological activity in accordance with the therapeutic methods of thepresent invention.

[0184] With respect to the therapeutic methods of the present invention,any subject can be treated, including animal and plant subjects. Apreferred subject is a vertebrate subject. Preferred examples ofvertebrates include fish and warm-blooded vertebrates. A preferredexample of a warm-blooded vertebrate is a mammal. A preferred example ofa mammal is a human. Additionally, as used herein and in the. Claims,the term “patient” is contemplated to include both human and animalpatients, and thus, veterinary therapeutic uses are contemplated inaccordance with the present invention.

[0185] Accordingly, the methods of the present invention areparticularly contemplated to be useful in the treatment of warm-bloodedvertebrates. Therefore, the invention concerns mammals and birds.

[0186] Contemplated is the treatment of mammals such as humans, as wellas those mammals of importance due to being endangered (such as Siberiantigers), of economical importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also contemplated is the treatment of birds, including thetreatment of those kinds of birds that are endangered, kept in zoos, aswell as fowl, and more particularly domesticated fowl, i.e., poultry,such as turkeys, chickens, ducks, geese, guinea fowl, and the like, asthey are also of economical importance to humans. Thus, contemplated isthe treatment of livestock, including, but not limited to, domesticatedswine (pigs and hogs), ruminants, horses, poultry, and the like.

Modulation of Piwi Family Biological Activity

[0187] In one embodiment, a therapeutic method according to the presentinvention comprises administering to a subject a substance that inhibitsor promotes piwi family biological activity. Such a substance may beidentified according to the screening assay set forth above. The methodcomprises treating a subject suffering from a disorder associated withor mediated by piwi family biological activity by administering to thesubject an effective piwi family biological activity-modulating amountof a substance identified according to a screening assay describedabove. By the term “modulating”, itis contemplated that the substancecan either promote or inhibit the biological activity of piwi familypolypeptides, depending on the disorder to be treated. For example, theadministering of antibodies against a chosen PIWI family protein (e.g.HIWI, MIWI, etc.) provides treatment of germline tumor or otherdevelopmental or tissue defects caused by piwi family-mediatedmechanisms.

[0188] Insofar as a piwi family biological activity modulator can takethe form of a polypeptide or of an anti-piwi family monoclonal antibody,or fragment thereof, it is to be appreciated that the potency, andtherefore an expression of a “therapeutically effective” amount canvary. However, as shown by the present assay methods, one skilled in theart can readily assess the potency of a candidate piwi family biologicalactivity modulator of this invention. A piwi family biological activitymodulator can be measured by a variety of means including through theuse of a responsive reporter, which drives expression of a reportergene; interaction of piwi family polypeptides with a monoclonal antibodyas described herein; and the like assays.

[0189] A preferred piwi family biological activity modulator has theability to substantially interact with piwi family in solution atmodulator concentrations of less than one (1)-micro molar (μM),preferably less than 0.1 μM, and more preferably less than 0.01 μM. By“substantially” is meant that at least a 50 percent modulation in piwifamily biological activity is observed in the presence of the piwifamily biological activity modulator, and a 50% modulation is referredto herein as an IC50 value.

[0190] A therapeutically effective amount of a piwi family biologicalactivity modulator of this invention in the form of a monoclonalantibody, or fragment thereof, is typically an amount such that whenadministered in a physiologically tolerable composition is sufficient toachieve a plasma concentration of from about 0.01 microgram (ug) permilliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml toabout 5 ug/ml, and usually about 5 ug/ml.

[0191] A therapeutically effective amount of a piwi family biologicalactivity modulator of this invention in the form of a polypeptide istypically an amount of polypeptide such that when administered in aphysiologically tolerable composition is sufficient to achieve a plasmaconcentration of from about 0.001 microgram (μg) per milliliter (ml) toabout 10 μg/ml, preferably from about 0.05 μg/ml to about 1.0 ug/ml.

[0192] The monoclonal antibodies or polypeptides of the invention can beadministered parenterally by injection or by gradual infusion over time.Although the tissue to be treated can typically be accessed in the bodyby systemic administration and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains the target molecule. Thus, monoclonalantibodies or polypeptides of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intra-cavity, transdermally, and can be delivered by peristaltic means.

[0193] The therapeutic compositions containing a monoclonal antibody ora polypeptide of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier or vehicle.

[0194] The compositions are administered in a manner compatible with thedosage formulation, and in a therapeutically effective amount. Thequantity to be administered depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgement of thepractitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are contemplated. Polypeptide modulatorscan also be modified as described herein below with respect tobiologically active piwi family proteins and protein therapy methodsusing the same.

Monoclonal Antibodies

[0195] The present invention describes, in one embodiment, piwi familymodulators in the form of monoclonal antibodies which immunoreact with apiwi family polypeptide and bind the piwi family polypeptide to modulatebiological activity as described herein. The invention also describesabove cell lines which produce the antibodies, methods for producing thecell lines, and methods for producing the monoclonal antibodies.

[0196] The term “antibody or antibody molecule” in the variousgrammatical forms is used herein as a collective noun that refers to apopulation of immunoglobulin molecules and/or immunologically active,portions of immunoglobulin molecules, i.e., molecules that contain anantibody combining site or paratope. An “antibody combining site” isthat structural portion of an antibody molecule comprised of heavy andlight chain variable and hypervariable regions that specifically bindsantigen.

[0197] Exemplary antibodies for use in the present invention are intactimmunoglobulin molecules, substantially intact immunoglobulin molecules,single chain immunoglobulins or antibodies, those portions of animmunoglobulin molecule that contain the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), and alsoreferred to as antibody fragments. Indeed, it is contemplated to bewithin the scope of the present invention that a monovalent modulatormay optionally be is used in the present method. Thus, the terms“modulate”, “modulating”, and “modulator” are meant to be construed toencompass such promotion.

[0198] The phrase “monoclonal antibody” in its various grammatical formsrefers to a population of antibody molecules that contain only onespecies of antibody combining site capable of immunoreacting with aparticular epitope. A monoclonal antibody thus typically displays asingle binding affinity for any epitope with which it immunoreacts. Amonoclonal antibody may therefore contain an antibody molecule having aplurality of antibody combining sites, each immunospecific for adifferent epitope, e.g., a bispecific monoclonal antibody. Methods ofproducing a monoclonal antibody, a hybridoma cell, or a hybridoma cellculture are described above.

[0199] It is also possible to determine, without undue experimentation,if a monoclonal antibody has the same (i.e., equivalent) specificity(immunoreaction characteristics) as a monoclonal antibody of thisinvention by ascertaining whether the former prevents the latter frombinding to a preselected target molecule. If the monoclonal antibodybeing tested competes with the monoclonal antibody of the invention, asshown by a decrease in binding by the monoclonal antibody of theinvention in standard competition assays for binding to the targetmolecule when present in the solid phase, then it is likely that the twomonoclonal antibodies bind to the same, or a closely related, epitope.

[0200] Still another way to determine whether a monoclonal antibody hasthe specificity Of a monoclonal antibody of the invention is topre-incubate the monoclonal antibody of the invention with the targetmolecule with which it is normally reactive, and then add the monoclonalantibody being tested to determine if the monoclonal antibody beingtested is inhibited in its ability to bind the target molecule. If themonoclonal antibody being tested is inhibited then, in all likelihood,it has the same, or functionally equivalent, epitopic specificity as themonoclonal antibody of the invention.

[0201] An additional way to determine whether a monoclonal antibody hasthe specificity of a monoclonal antibody of the invention is todetermine the amino acid residue sequence of the CDR regions of theantibodies in question. “CDRs” (complementarity determining regions)mean the three subregions of the light or heavy chain variable regionswhich have hypervariable sequences and form loop structures that areprimarily responsible for making direct contact with antigen. Antibodymolecules having identical, or functionally equivalent, amino acidresidue sequences in their CDR regions have the same bindingspecificity. Methods for sequencing polypeptides are well known in theart.

[0202] The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part bythe, amino acid residue sequence of the variable region of the heavychain of the immunoglobulin that comprises the antibody, and in part bythe light chain variable region amino acid residue sequence. Use of theterms “having the binding specificity of” or “having the bindingpreference of” indicates that equivalent monoclonal antibodies exhibitthe same or similar immunoreaction (binding) characteristics and competefor binding to a preselected target molecule.

[0203] Humanized monoclonal antibodies offer particular advantages overmurine monoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies. Methods of preparing “humanized” antibodies aregenerally well known in the art, and can readily be applied to theantibodies of the present invention. Thus, the invention contemplates,in one embodiment, a monoclonal antibody of this invention that ishumanized by grafting to introduce components of the human immune systemwithout substantially interfering with the ability of the antibody tobind antigen.

[0204] The use of a molecular cloning approach to generate antibodies,particularly monoclonal antibodies, and more particularly single chainmonoclonal antibodies, is also contemplated. The production of singlechain antibodies has been described in the art, see e.g., U.S. Pat. No.5,260,203, the contents of which are herein incorporated by reference.For this, combinatorial immunoglobulin phagemid libraries are preparedfrom RNA isolated from the spleen of the immunized animal, and phagemidsexpressing appropriate antibodies are selected by panning on endothelialtissue. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination in a single chain, which furtherincreases the chance of finding appropriate antibodies. Thus, anantibody of the present invention, or a “derivative” of an antibody ofthe present invention pertains to a single polypeptide chain bindingmolecule which has binding specificity and affinity substantiallysimilar to the binding specificity and affinity of the light and heavychain aggregate variable region of an antibody described herein.

Other Modulators

[0205] Given the disclosure of the piwi family activity in tissuesherein, it is also contemplated that as yet undefined chemical compoundsmay be used to modulate piwi family activity in tissues in accordancewith the methods of the present invention. The identification of suchcompounds is facilitated by the description of screening assays directedto piwi family activity in tissues presented above.

Gene Therapy

[0206] In accordance with the present invention, piwi family genes canbe used as a tool of gene therapy in humans and mammalian, avian, andfish systems to increase the number of stem cells and to increaseproliferation of primordial germ cells in vivo.

[0207] For example, over-proliferation of malignant stem cells is theleading cause of cancer while under-proliferation of stem cells orstem-like progenitor cells leads to tissue dystrophy, anemia,immunodeficiency, and male infertility. The crucial role of stem cellshas long been attributed to their ability to self-renew and to generateimmense number of specialized cells on demand. Thus, piwi family genescan be used for gene therapy in accordance with the present invention.Exemplary gene therapy methods, including liposomal transfection ofnucleic acids into host cells, are described in U.S. Pat. Nos.5,279,833; 5,286,634; 5,399,346; 5,646,008; 5,651,964; 5,641,484; and5,643,567, the contents of each of which are herein incorporated byreference.

[0208] Briefly, piwi family gene therapy directed toward modulation ofgrowth, proliferation and self-renewing division in a target cell isdescribed. Target cells include but are not limited to germline stemcells, primordial germ cells and cancerous or tumorous cells. In oneembodiment, a therapeutic method of the present invention contemplates amethod for modulating of growth, proliferation or self-renewing divisionin a cell comprising the steps of: (a) delivering to the cell aneffective amount of a DNA molecule comprising a polynucleotide thatencodes a piwi family polypeptide that modulates growth, proliferationor self-renewing division; and (b) maintaining the cell under conditionssufficient for expression of said polypeptide.

[0209] In a preferred embodiment, the piwi family polypeptide is HIWI orMIWI. Delivery is preferably accomplished by injecting the DNA moleculeinto the cell. Where the cell is in a subject delivering is preferablyadministering the DNA molecule into the circulatory system of thesubject. In a preferred embodiment, administering comprises the stepsof: (a) providing a vehicle that contains the DNA molecule; and (b)administering the vehicle to the subject.

[0210] A vehicle is preferably a cell transformed or transfected withthe DNA molecule or a transfected cell derived from such a transformedor transfected cell. An exemplary and preferred transformed ortransfected cell is a spermatogonial cell or a tumor cell from the tumorbeing treated. Techniques for transforming or transfecting a cell with aDNA molecule of the present invention are set forth above.

[0211] Alternatively, the vehicle is a virus or an antibody thatspecifically infects or immunoreacts with an antigen of the tumor.Retroviruses used to deliver the constructs to the host target tissuesgenerally are viruses in which the 3′-LTR (linear transfer region) hasbeen inactivated. That is, these are enhancerless 3′-LTR's, oftenreferred to as SIN (self-inactivating viruses) because after productiveinfection into the host cell, the 3′-LTR is transferred to the 5′-endand both viral LTR's are inactive with respect to transcriptionalactivity. A use of these viruses well known to those skilled in the artis to clone genes for which the regulatory elements of the cloned geneare inserted in the space between the two LTR's. An advantage of a viralinfection system is that it allows for a very high level of infectioninto the appropriate recipient cell.

[0212] Antibodies have been used to target and deliver DNA molecules. AnN-terminal modified poly-L-lysine (NPLL)-antibody conjugate readilyforms a complex with plasmid DNA. A complex of monoclonal antibodiesagainst a cell surface thrombomodulin conjugated with NPLL was used totarget a foreign plasmid DNA to an antigen-expressing mouse lungendothelial cell line and mouse lung. Those targeted endothelial cellsexpressed the product encoded by that foreign DNA.

[0213] It is also envisioned that this embodiment of the presentinvention can be practiced using alternative viral or phage vectors,including retroviral vectors, adenoviral vectors and vaccinia viruseswhose genome has been manipulated in alternative ways so as to renderthe virus non-pathogenic. Methods for creating such a viral mutation areset forth in detail in U.S. Pat. No. 4,769,331, incorporated herein byreference.

[0214] By way of specific example, hiwi, miwi, and their homologs fromwarm-blooded vertebrates are introduced into isolated spermatogonialcells or other relevant cells. The re-injection of thetransgene-carrying cells into the testis or other relevant tissuesprovides a treatment for male infertility or other relevant diseases inhuman and animals.

Protein Therapy

[0215] In another embodiment, the direct introduction of the piwi familyproteins into a diseased tissue is contemplated to provide a therapeuticeffect in human and animals. Such a therapeutic method comprisesadministering to a subject a therapeutic composition which comprises abiologically active piwi family polypeptide of the present invention inamount effective to modulate a piwi− mediated biological activity in thesubject.

[0216] In one embodiment, a polypeptide for use in such a compositioncomprises no more than about 100 amino acid residues, preferably no morethan about 60 residues, more preferably no more than about 30 residues.Peptides can be linear or cyclic.

[0217] A subject polypeptide includes any analog, fragment or chemicalderivative of a piwi family polypeptide. Such a polypeptide can besubject to various changes, substitutions, insertions, and deletionswhere such changes provide for certain advantages in its use. In thisregard, an piwi family polypeptide for use in a therapeutic method ofthe present invention corresponds to, rather than is identical to, thesequence of a native piwi family polypeptide where one or more changesare made and it retains the ability to function as a piwi familypolypeptide in one or more of the assays as defined herein. Thus, apolypeptide can be in any of a variety of forms of peptide derivatives,that include amides, conjugates with proteins, cyclized peptides,polymerized peptides, analogs, fragments, chemically modified peptides,and the like derivatives.

[0218] The term “analog” includes any polypeptide having an amino acidresidue sequence substantially identical to a sequence of an endogenouspiwi family polypeptide in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the piwi family biological activity as described herein.Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another; the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine; the substitutionof one basic residue such as lysine, arginine or histidine for another;or the substitution of one acidic residue, such as aspartic acid orglutamic acid for another. Such substitutions are described in detailabove with respect to the isolated and purified piwi family polypeptideof the present invention.

[0219] The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite biologicalactivity.

[0220] “Chemical derivative” refers to a subject polypeptide having oneor more residues chemically derivatized by reaction of a functional sidegroup. Such derivatized, molecules include for example, those moleculesin which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For examples: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

[0221] The term fragment” refers to any subject polypeptide having anamino acid residue sequence shorter than that of a polypeptide whoseamino acid residue sequence is shown herein.

[0222] When a polypeptide of the present invention has a sequence thatis not identical to the sequence of a piwi family endogenouspolypeptide, it is typically because one or more conservative ornon-conservative substitutions have been made, usually no more thanabout 30 number percent, and preferably no more than 10 number percentof the amino acid residues are substituted. Additional residues may alsobe added at either terminus of a polypeptide for the purpose ofproviding a “linker” by which the polypeptides of this invention can beconveniently affixed to a label or solid matrix, or carrier. Labels,solid matrices and carriers that can be used with the polypeptides ofthis invention are described elsewhere herein.

[0223] Amino acid residue linkers are usually at least one residue andcan be 40 or more residues, more often 1 to 10 residues, but do not formpiwi family polypeptide epitopes. Typical amino acid residues used forlinking are tyrosine, cysteine, lysine, glutamic and aspartic acid, orthe like. In addition, a subject polypeptide can differ, unlessotherwise specified, from the natural sequence of a piwi familypolypeptide by the sequence being modified by terminal-NH2 acylation,e.g., acetylation, or thioglycolic acid amidation, byterminal-carboxylamidation, e.g., with ammonia, methylamine, and thelike terminal modifications. Terminal modifications are useful, as iswell known, to reduce susceptibility by proteinase digestion, andtherefore serve to prolong half life of the polypeptides in solutions,particularly biological fluids where proteases may be present. In thisregard, polypeptide cyclization is also a useful terminal modification,and is particularly preferred also because of the stable structuresformed by cyclization and in view of the biological activities observedfor such cyclic peptides as described herein.

[0224] Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable ofthe peptides with the peptides of the present invention includeinorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalenesulfonic acid, sulfanilic acid or the like. HCl and TFA salts areparticularly preferred.

[0225] Suitable bases capable of forming salts with the peptides of thepresent invention include inorganic bases such as sodium hydroxide,ammonium hydroxide, potassium hydroxide and the like; and organic basessuch as mono- di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like), andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

[0226] A peptide of the present invention, also referred to herein as asubject polypeptide, can be synthesized by any of the techniques thatare known to those skilled in the polypeptide art, including recombinantDNA techniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in Steward et al., “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San-Francisco, 1969; Bodanszky, et al.,“Peptide Synthesis”, John. Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York),1983; Merrifield, Adv Enzymol, 32:221-96,1969; Fieldset al., Int. J. Peptide Protein Res., 35:161-214,1990; and U.S. Pat. No.4,244,946 for solid phase peptide synthesis, and Schroder et al., ‘ThePeptides’, Vol. 1, Academic Press (New York), 1965 for classicalsolution synthesis, each of which is incorporated herein by reference.Appropriate protective groups usable in such synthesis are described inthe above texts and in J. F. W. McOmie, “Protective Groups in OrganicChemistry”, Plenum Press, New York, 1973, which is incorporated hereinby reference.

[0227] In general, the solid-phase synthesis methods contemplatedcomprise the sequential addition of one or more amino acid residues orsuitably protected amino acid residues to a growing peptide chain.Normally, either the amino or carboxyl group of the first amino acidresidue is protected by a suitable, selectively removable protectinggroup a different, selectively removable protecting group is utilizedfor amino acids containing a reactive side group such as lysine.

[0228] Using a solid phase synthesis as exemplary, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

[0229] The resultant linear polypeptides prepared for example asdescribed above may be reacted to form their corresponding cyclicpeptides. An exemplary method for cyclizing peptides is described byZimmer et al., Peptides 1992, pp. 393-394, ESCOM Science Publishers, B.V., 1993. Typically, tertbutoxycarbonyl protected peptide methyl esteris dissolved in methanol and sodium hydroxide solution are added and theadmixture is reacted at 20° C. to hydrolytically remove the methyl esterprotecting group. After evaporating the solvent, the tertbutoxycarbonylprotected peptide is extracted with ethyl acetate from acidified aqueoussolvent. The tertbutoxycarbonyl protecting group is then removed undermildly acidic conditions in dioxane cosolvent. The unprotected linearpeptide with free amino and carboxy termini so obtained is converted toits corresponding cyclic peptide by reacting a dilute solution of thelinear peptide, in a mixture of dichloromethane and dimethylformamide,with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazoleand N-methylmorpholine. The resultant cyclic peptide is then purified bychromatography.

Antisense Oligonucleotide Therapy

[0230] In another embodiment, the administering of antisense RNA againsta chosen piwi family gene (e.g. hiwi, miwi, etc.) provides treatment ofgermline tumor or other developmental or tissue defects caused by piwifamily-mediated mechanisms. In accordance with the present inventionsuch a therapeutic method may alternatively comprise promoting orinhibiting piwi family in a subject by administering an effective amountof a substance that inhibits or promotes expression of a piwifamily-encoding nucleic acid segment in the subject. Examples of such asubstance, include, for example, an antisense oligonucleotide derivedfrom SEQ. ID NOs:1, 3 or 5. Therapeutic methods utilizing antisenseoligonucleotides have been described in the art, for example in U.S.Pat. Nos. 5,627,158 and 5,734,033, the contents of each of which areherein incorporated by reference.

Formulation of Therapeutic Compositions

[0231] The piwi family biological activity modulating substances, genetherapy vectors, biologically activity piwi family gene products, andsubstances that inhibit or promote expression of a piwi family-encodingnucleic acid segment described above are adapted for administration as apharmaceutical compositions as described above. Additional formulationand dose preparation techniques have been described in the art, see forexample, those described in U.S. Pat. No. 5,326,902 issued to Seipp etal. on Jul. 5, 1994, U.S. Pat. No. 5,234,933 issued to Marnett et al. onAug. 10, 1993, and PCT Publication WO 93/25521 of Johnson et al.published Dec. 23, 1993, the entire contents of each of which are hereinincorporated by reference.

[0232] For the purposes described above, the identified substances maynormally be administered systemically or partially, usually by oral orparenteral administration. The doses to be administered are determineddepending upon age, body weight, symptom, the desired therapeuticeffect, the route of administration, and the duration of the treatmentetc. In a human adult, the doses per person per administration aregenerally between 1 mg and 500 mg, by oral administration, up to severaltimes per day, and between 1 mg and 100 mg, by parenteral administrationup to several times per day. Since the doses to be used depend uponvarious conditions, as mentioned above, there may be a case in whichdoses are lower than or greater than the ranges specified above.

[0233] Solid compositions for oral administration include compressedtablets, pills, dispersible powders, capsules, and granules. In suchcompositions, one or more of the active substance(s) is or are, admixedwith at least one inert diluent (lactose, mannitol, glucose,hydroxypropylcellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate alminate, etc.). Thecompositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (magnesiumstearate, etc.), disintegrating agents (cellulose, calcium glycolateetc.), and assisting agent for dissolving (glutamic acid, aspartic acid,etc.) stabilizing agent (lactose etc.). The tablets or pills may, ifdesired, be coated with gastric or enteric material (sugar, gelatin,hydroxypropylcellulose or hydroxypropylmethyl cellulose phthalate,etc.). Capsules include soft ones and hard ones.

[0234] Liquid compositions for oral administration includepharmaceutically-acceptable emulsions, solutions, suspensions, syrupsand elixirs. In such compositions, one or more of the activesubstance(s) is or are admixed with inert diluent(s) commonly used inthe art (purified water, ethanol etc.). Besides inert diluents, suchcompositions may also comprise adjuvants (wetting agents, suspendingagents, etc.), sweetening agents, flavoring agents, perfuming agents andpreserving agents.

[0235] Other compositions for oral administration include spraycompositions which may be prepared by known methods and which compriseone or more of the active substance(s). Spray compositions may compriseadditional substances other than inert diluents: e.g. preserving agents(sodium sulfite, etc.), isotonic buffer (sodium chloride, sodiumcitrate, citric acid, etc.). For preparation of such spray compositions,for example, the method described in U.S. Pat. No. 2,868,691 or3,095,355 may be used.

[0236] Injections for parenteral administration include sterile aqueousor non-aqueous solution, suspensions and emulsions. In suchcompositions, one or more of active substance(s) is or are admixed withat least one inert aqueous diluent(s) (distilled water for injection,physiological salt solution etc.) or inert non-aqueous diluent(s)(propylene glycol, polyethylene glycol, olive oil, ethanol, POLYSOLBATE80® etc.). Injections may comprise additional other than inert diluents:e.g. preserving agents, wetting agents, emulsifying agents, dispersingagents, stabilizing agents (lactose, etc.), assisting agents such as fordissolving (glutamic acid, aspartic acid, etc.). They may be sterilized,for example, by filtration through a bacteria-retaining filter, byincorporation of sterilizing agents in the compositions or byirradiation. They also be manufactured in the form of sterile solidcompositions, for example, by freeze-drying, and which can be dissolvedin sterile water or some other sterile diluents for injectionimmediately before use.

[0237] Other compositions for administration include liquids forexternal use, and endermic linaments (ointment, etc.), suppositories andpessaries which comprise one or more of the active substance(s) and maybe prepared by known methods.

Detecting a Polynucleotide or a Polypeptide of the Present Invention

[0238] Alternatively, the present invention provides a method ofdetecting a polypeptide of the present invention, wherein the methodcomprises immunoreacting the polypeptides with antibodies preparedaccording to the method described above to form antibody-polypeptideconjugates, and detecting the conjugates.

[0239] In yet another embodiment, the present invention contemplates amethod of detecting messenger RNA transcripts that encode a polypeptideof the present invention, wherein the method comprises hybridizing themessenger RNA transcripts with polynucleotide sequences that encode thepolypeptide to form duplexes; and detecting the duplex. Alternatively,the present invention provides a method of detecting DNA molecules thatencode a polypeptide of the present invention, wherein the methodcomprises hybridizing DNA molecules with a polynucleotide that encodesthat polypeptide to form duplexes; and detecting the duplexes.

[0240] The detection and screening assays disclosed herein can be usedas a prognosis tool hiwi, miwi and their mammalian homologous genes aswell as their protein products can be readily used in clinical settingas a prognostic indicator for screening infertility, germline cancer, orother heritable piwi-related diseases in human and animals.

[0241] The detection and screening assays disclosed-herein can be alsoused as a part of a diagnostic method. hiwi, miwi, and their mammalianhomologs as well as their proteins can be readily used in clinicalpractice to diagnose the cause of infertility, testicular cancer, orother heritable piwi-related diseases in human and animals.

Screening Assays for a Polypeptide of the Present Invention

[0242] The present invention provides a method of screening a biologicalsample for the presence of a piwi family polypeptide. Preferably, thepiwi family polypeptide possesses a biological activity in accordancewith the present invention. A biological sample to be screened can be abiological fluid such as extracellular or intracellular fluid or a cellor tissue extract or homogenate. A biological sample can also be anisolated cell (e.g., in culture) or a collection of cells such as in atissue sample or histology sample. A tissue sample can be suspended in aliquid medium or fixed onto a solid support such as a microscope slide.

[0243] In accordance with a screening assay method, a biological sampleis exposed to an antibody immunoreactive with the polypeptide whosepresence is being assayed. Typically, exposure is accomplished byforming an admixture in a liquid medium that contains both the antibodyand the candidate polypeptide. Either the antibody or the sample withthe polypeptide can be affixed to a solid support (e.g., a column or amicrotiter plate).

[0244] The biological sample is exposed to the antibody under biologicalreaction conditions and for a period of time sufficient forantibody-polypeptide conjugate formation. Biological reaction conditionsinclude ionic composition and concentration, temperature, pH and thelike.

[0245] Ionic composition and concentration can range from that ofdistilled water to a 2 molal solution of NaCl. Preferably, osmolality isfrom about 100 mosmols/I to about 400 mosmols/I and, more preferablyfrom about 200 mosmols/l to about 300 mosmols/I. Temperature preferablyis from about 4° C. to about 100° C., more preferably from about 15° C.to about 500C. and, even more preferably from about 25° C. to about 40°C. pH is preferably from about a value of 4.0 to a value of about 9.0,more preferably from about a value of 6.5 to a value of about 8.5 and,even more preferably from about a value of 7.0 to a value of about 7.5.The only limit on biological reaction conditions is that the conditionsselected allow for antibody-polypeptide conjugate formation and that theconditions do not adversely affect either the antibody or thepolypeptide.

[0246] Exposure time will vary inter alia with the biological conditionsused, the concentration of antibody and polypeptide and the nature ofthe sample (e.g., fluid or tissue sample). Means for determiningexposure time are well known to one of ordinary skill in the art.Typically, where the sample is fluid and the concentration ofpolypeptide in that sample is about 10⁻¹⁰M, exposure time is from about10 minutes to about 200 minutes.

[0247] The presence of polypeptide in the sample is detected bydetecting the formation and presence of antibody-polypeptide conjugates.Means for detecting such antibody-antigen (e.g., receptor polypeptide)conjugates or complexes are well known in the art and include suchprocedures as centrifugation, affinity chromatography and the like,binding of a secondary antibody to the antibody-candidate receptorcomplex.

[0248] In one embodiment, detection is accomplished by detecting anindicator affixed to the antibody. Exemplary and well known suchindicators include radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a secondantibody or an enzyme such as horse radish peroxidase. Means foraffixing indicators to antibodies are well known in the art. Commercialkits are available.

Screening Assay for Anti-Polypeptide Antibody

[0249] In another aspect, the present invention provides a method ofscreening a biological sample for the presence of antibodiesimmunoreactive with a piwi family polypeptide. Preferably the piwifamily polypeptide has a biological activity in accordance with thepresent invention. In accordance with such a method, a biological sampleis exposed to a piwi family polypeptide under biological conditions andfor a period of time sufficient for antibody-polypeptide conjugateformation and the formed conjugates are detected.

Screening Assay for Polynucleotide That Encodes a piwi FamilyPolypeptide of the Present Invention

[0250] A DNA molecule and, particularly a probe molecule, can be usedfor hybridizing as an oligonucleotide probe to a DNA source suspected ofencoding a piwi family polypeptide of the present invention. Preferablythe piwi family polypeptide possesses a biological activity inaccordance with the present invention. The probing is usuallyaccomplished by hybridizing the oligonucleotide to a DNA sourcesuspected of possessing a piwi family gene. In some cases, the probesconstitute only a single probe, and in others, the probes constitute acollection of probes based on a certain amino acid sequence or sequencesof the polypeptide and account in their diversity for the redundancyinherent in the genetic code.

[0251] A suitable source of DNA for probing in this manner is capable ofexpressing a polypeptide of the present invention and can be a genomiclibrary of a cell line of interest. Alternatively, a source of DNA caninclude total DNA from the cell line of interest. Once the hybridizationmethod of the invention has identified a candidate DNA segment, oneconfirms that a positive clone has been obtained by furtherhybridization, restriction enzyme mapping, sequencing and/or expressionand testing.

[0252] Alternatively, such DNA molecules can be used in a number oftechniques including their use as: (1) diagnostic tools to detect normaland abnormal DNA sequences in DNA derived from patient's cells; (2)reagents for detecting and isolating other members of the polypeptidefamily and related polypeptides from a DNA library potentiallycontaining such sequences; (3) primers for hybridizing to relatedsequences for the purpose of amplifying those sequences; (4) primers foraltering native piwi family DNA sequences; as well as other techniqueswhich rely on the similarity of the DNA sequences to those of the DNAsegments herein disclosed.

[0253] As set forth above, in certain aspects, DNA sequence informationprovided by the invention allows for the preparation of relatively shortDNA (or RNA) sequences (e.g., probes) that specifically hybridize toencoding sequences of a selected piwi family gene. In these aspects,nucleic acid probes of an appropriate length are prepared based on aconsideration of the encoding sequence for a polypeptide of thisinvention. The ability of such nucleic acid probes to specificallyhybridize to other encoding sequences lend them particular utility in avariety of embodiments. Most importantly, the probes can be used in avariety of assays for detecting the presence of complementary sequencesin a given sample. However, other uses are envisioned, including the useof the sequence information for the preparation of mutant speciesprimers, or primers for use in preparing other genetic constructions.

[0254] To provide certain of the advantages in accordance with theinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes probe sequences that are complementary to atleast a 14 to 40 or so long nucleotide stretch of a nucleic acidsequence of the present invention, such as a sequence shown in any ofSEQ ID NOs:1, 3 and 5. A size of at least 14 nucleotides in length helpsto ensure that the fragment is of sufficient length to form a duplexmolecule that is both stable and selective. Molecules havingcomplementary sequences over stretches greater than 14 bases in lengthare generally preferred, though, to increase stability and selectivityof the hybrid, and thereby improve the quality and degree of specifichybrid molecules obtained. One will generally prefer to design nucleicacid molecules having gene-complementary stretches of 14 to 20nucleotides, or even longer where desired. Such fragments can be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, by application of nucleic acid reproduction technology, such asthe PCR technology of U.S. Pat. No. 4,683,202, herein incorporated byreference, or by introducing selected sequences into recombinant vectorsfor recombinant production.

[0255] Accordingly, a nucleotide sequence of the present invention canbe used for its ability to selectively form duplex molecules withcomplementary stretches of the gene. Depending on the applicationenvisioned, one employs varying conditions of hybridization to achievevarying degrees of selectivity of the probe toward the target sequence.For applications requiring a high degree of selectivity, one typicallyemploys relatively stringent conditions to form the hybrids. Forexample, one selects relatively low salt and/or high temperatureconditions, such as provided by 0.02M-0.15M NaCl at temperatures of 50°C. to 70° C. Such conditions are particularly selective, and toleratelittle, if any, mismatch between the probe and the template or targetstrand.

[0256] Of course, for some applications, for example, where one desiresto prepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate polypeptide codingsequences from related species, functional equivalents, or the like,less stringent hybridization conditions are typically needed to allowformation of the heteroduplex. Under such circumstances, one employsconditions such as 0.15M-0.9M salt, at temperatures ranging from 20° C.to 55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

[0257] In certain embodiments, it is advantageous to employ a nucleicacid sequence of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Inpreferred embodiments, one likely employs an enzyme tag such a urease,alkaline phosphatase or peroxidase, instead of radioactive or otherenvironmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known which can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

[0258] In general, it is envisioned that the hybridization probesdescribed herein are useful both as reagents in solution hybridizationas well as in embodiments employing a solid phase. In embodimentsinvolving a solid phase, the sample containing test DNA (or RNA) isadsorbed or otherwise affixed to a selected matrix or surface. Thisfixed, single-stranded nucleic acid is then subjected to specifichybridization with selected probes under desired conditions. Theselected conditions depend inter alia on the particular circumstancesbased on the particular criteria required (depending, for example, onthe G+C contents, type of target nucleic acid, source of nucleic acid,size of hybridization probe, etc.). Following washing of the hybridizedsurface so as to remove nonspecifically bound probe molecules, specifichybridization is detected, or even quantified, by means of the label.

Assay Kits

[0259] In another aspect, the present invention contemplates assay kitsfor detecting the presence of a polypeptide of the present invention inbiological samples, where the kits comprise a first container containinga first antibody capable of immunoreacting with the polypeptide, withthe first antibody present in an amount sufficient to perform at leastone assay. Preferably, the assay kits of the invention further comprisea second container containing a second antibody that immunoreacts withthe first antibody. More preferably, the antibodies used in the assaykits of the present invention are monoclonal antibodies. Even morepreferably, the first antibody is affixed to a solid support. Morepreferably still, the first and second antibodies comprise an indicator,and, preferably, the indicator is a radioactive label or an enzyme.

[0260] The present invention also contemplates a kit for screeningagents. Such a kit can contain a polypeptide of the present invention.The kit can contain reagents for detecting an interaction between anagent and a receptor of the present invention. The provided reagent canbe radiolabeled. The kit can contain a known radiolabelled agent capableof binding or interacting with a receptor of the present invention.

[0261] In an alternative aspect, the present invention provides assaykits for detecting the presence, in biological samples, of apolynucleotide that encodes a polypeptide of the present invention, thekits comprising a first container that contains a second polynucleotideidentical or complementary to a segment of at least 10 contiguousnucleotide bases of, as a preferred example, SEQ ID NOs:1, 3 and 5.

[0262] In another embodiment, the present invention contemplates assaykits for detecting the presence, in a biological sample, of antibodiesimmunoreactive with a polypeptide of the present invention, the kitscomprising a first container containing a piwi family polypeptide, thatimmunoreacts with the antibodies, with the polypeptide present in anamount sufficient to perform at least one assay. Preferably, the piwifamily polypeptide has a biological activity in accordance with thepresent invention. The reagents of the kit can be provided as a, liquidsolution, attached to a solid support or as a dried powder. Preferably,when the reagent is provided in a liquid solution, the liquid solutionis an aqueous solution. Preferably, when the reagent provided isattached to a solid support, the solid support can be chromatographmedia or a microscope slide. When the reagent provided is a dry powder,the powder can be reconstituted by the addition of a suitable solvent.The solvent can be provided.

EXAMPLES

[0263] The following examples have been included to illustrate preferredmodes of the invention. Certain aspects of the following examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

[0264] In exploring stem cell renewal mechanisms in the germline, thepiwi gene is disclosed herein to be essential for GSC maintenance inDrosophila. The role of the piwi gene is characterized herein byrecessive mutations that cause failure in GSC maintenance in bothfemales and males. piwi mutant gonads contain a normal number of GSCs atthe onset of gametogenesis. However, mutant adult gonads are devoid ofGSCs and contain only a very small number of gametes that isapproximately equal to or less than the wild-type number of GSCs. Theseobservations reveal that piwi is essential for GSC maintenance in bothmales and females.

Summary of Observations in Examples

[0265] The purification and isolation of cDNAs representing piwi familygenes from human, mouse, and Drosophila are disclosed in the Examples,along with the primary sequence of the PIWI proteins in human, mouse andDrosophila as deduced from the isolated cDNA sequences.

[0266] Further, the identification of piwi homologous genes, prg-I andprg-2, and their role in germline self-renewal in C. elegans isdescribed in the Examples. The identification of two piwi family-likegenes in Arabidopsis, Zwille and argonaute(ago), that are required formeristem cell divisions is also described.

[0267] A highly conserved protein signature motif of 43-amino acidresidues, the PIWI box, that exists in piwi family genes from human,mouse, Drosophila, C. elegans, and Arabidopsis, as well as in a broaderclass of novel genes is also described in the Examples.

[0268] The demonstration that the PIWI and MIWI proteins are nuclearproteins is also disclosed.

[0269] The essential function of piwi for Drosophila germline stem cellgrowth, proliferation and self-renewal and also for early embryonicdevelopment is also disclosed.

[0270] The specific expression pattern of miwi and hiwi in the adultmurine and human testis, respectively, but not in the adult murine orhuman ovary which do not contain germline stem cells is alsodemonstrated in the Examples. miwi is further shown to be expressed ingermline stem cells (and specifically not in somatic cells) of the mousetestis.

[0271] The essential role of piwi family genes in mammalianspermatogenesis is also disclosed. A null mutation of miwi, wherein mostof the miwi sequence is deleted, results in male infertility withspecific defects during early stages of spermatogenesis. A mutation thatspecifically deletes miwi sequences encoding the PIWI box showabnormalities indistinguishable from those of the null mutant,demonstrating the essentiality of the PIWI box for piwi gene functions.

[0272] Also disclosed is a demonstration that miwi contributes to thelong term self-renewing ability of hematopoietic stem cells.

[0273] Thus, as disclosed herein, the function of piwi family genes inregulating proliferation of germ cells and other stem cells is conservedamong human, mouse, Drosophila, C. elegans, and Arabidopsis.

Example 1 Isolation and Purification of piwi

[0274] The cloning and characterization of the Drosophila piwi gene isdisclosed in this Example. Germline stem cells (GSCs) serve as thesource for gametogenesis in diverse organisms. The piwi gene product isrequired for the asymmetric division of GSC to produce and maintain adaughter GSC but is not essential for the further differentiation of thecommitted daughter cell. Genetic mosaic and RNA in situ analysesindicate that piwi expression in adjacent somatic cells regulates GSCdivision.

[0275] piwi encodes a highly basic novel protein well conserved duringevolution. This Example also describes the isolation of piwi familyhomologs in C. elegans and human. This Example further describes theidentification of Arabidopsis piwi family-like genes known to berequired for meristem cell maintenance. Decreasing C. elegans piwiexpression reduces the proliferation of GSC-equivalent cells. Thus, piwirepresents a novel class of genes required for GSC division in diverseorganisms.

[0276]Drosophila has been used in the art as a model for studyingmechanisms that are conserved among diverse developmental systems. Asshow herein, this is also the case for the study of stem cells. InDrosophila, stem cells exist in the germline at the apical tip of theovariole, the functional unit of the ovary. In the ovariole, germlinestem cells (GSCs) are located in a specialized structure called thegermarium, as first proposed by Brown and King (1962, 1964). Theexistence of GSCs was strongly supported by germline clonal analysis(Schüpbach et al., 1978; Wieschaus and Szabad, 1979) and directlyverified by laser ablation (Lin and Spradling, 1993). In each germarium,2-3 GSCs contact the somatic basal terminal filament cells. GSCs undergooriented asymmetric divisions to produce a daughter stem cell whichremains associated with the terminal filament and a differentiateddaughter, the cystoblast; that becomes displaced one cell away from theterminal filament (Lin and Spradling, 1997; Deng and Lin, 1997). Thegermline cyst then becomes enveloped by follicle cells produced bysomatic stem cells (Margolis and Spradling, 1995) to form an eggchamber, which buds off the germarium, joins pre-existing egg chambersin a linear array to form an ovariole, and eventually develops intoarmature egg (reviewed in King, 1970; Spradling, 1993). Thisassembly-line organization, with each egg chamber representing adifferentiated stem cell product whose position along the ovariolecorresponds to its birth order, provides a unique opportunity to studystem cell division.

[0277] To investigate the nature of the piwi-mediated mechanism, thisExample reports that piwi mediates a somatic signaling mechanismessential for the division and maintenance of GSCs in Drosophila.Moreover, piwi encodes a novel basic protein well conserved in C.elegans and humans, and also conserved in Arabidopsis, where two piwifamily-like genes are known to be required for meristem cellmaintenance. It is also demonstrated that the C. elegans piwi familyhomolog is also required for germline proliferation and maintenance.Thus, piwi family genes represent essential stem cell genes existing indiverse organisms.

[0278]Drosophila strains and culture. All strains were grown at 25° C.on yeast-containing molasses/agar medium. The piwi¹, piwi³ and piwi⁴mutations are single PZ insertional mutations reported by Lin andSpradling (1997) while the piwi² mutation is a single P-ry¹¹transposable insertion. Oregon R (Ore-R) serves as the wildtype strainfor all experiments.

[0279] Phenotypic analysis of piwi mutants. Piwi mutations were balancedover CyO-y⁺ and introduced into the y/y genetic background. Thehomozygous and heterozygous larvae were separated at the late thirdinstar stage by scoring mouth hooks and denticle belts for y. They werethen dissected to obtain the ovary immediately or were aged at 25° C. todesired pupal stages before dissection. Dissected ovaries were analyzedby immunofluorescence microscopy for defects in ovary differentiationand GSC division.

[0280] Immunocytochemistry and immunofluorescence microscopy. Wild typeand mutant ovaries from larval, pupal, or adult stages were dissected,fixed and stained as described by Lin et al. (1994). Forimmunofluorescence staining, anti-VASA antibodies (Hay et al., 1990)were used to specifically mark germ cells at 1:2000 dilution.Anti-α-spectrin antibodies (Byers et al., 1987) were used to outlinesomatic cells and to mark spectrosomes and fusomes (Lin and Spradling,1995) at 1:200 dilution. The monoclonal anti-MYC epitope antibody1-9E10.2 was described by Evan et al. (1985) and was used at 1:2dilution. All the fluorescence-conjugated secondary antibodies were fromJackson ImmunoResearch Laboratory Inc., Westgrove, Pa., and were used at1:200 dilution.

[0281] Immunofluorescently labeled samples were also counter-stainedwith the DNA specific dye DAPI as described in Lin and Spradling (1993).The immunologically labeled samples were examined using Nomarski andepifluorescence microscopy under a Zeiss AXIOPLAN® microscope (Zeiss,Oberkochen, Germany) equipped with a STAR-1™ cooled CCD camera(Photometrics, Tucson, Ariz.). Selected samples were further analyzed byconfocal microscopy using a Zeiss LSM410™ confocal microscope mounted onan AXIOVERT® 100. Images from the Zeiss AXIOPLAN® were collected usingIP Lab software and confocal images were processed using the LSM410software. All images were processed using the Adobe Photoshop™ program(Adobe Systems, Inc., San Jose, Calif.).

[0282] Cloning of piwi and mapping of its mutations. piwi maps to theleft arm of the second chromosome within polytene band 32C, as definedby insertions of single transposable elements (Lin and Spradling, 1997).DNA fragments flanking the piwi¹, piwi³, and piwi⁴ insertions wererecovered by a plasmid rescue method (Wilson. et al., 1989). Genomic DNAin the rescued plasmid was used to isolate additional genomic clonesfrom a λ genomic DNA library (Maniatis et al., 1978) to constitute a 36kb region of overlapping walk clones (FIG. 2). The insertion sites forpiwi¹, piwi³, and piwi⁴ were first mapped by Southern blots of EcoRIdigested walk clones, and then precisely determined by DNA sequencing.The site of the piwi² insertion was determined by sequencing itsflanking DNA fragments isolated by inverse PCR of piwi² genomic DNAusing primers specific to the P-element termini (Ochman et al., 1990).

[0283] Genomic DNA fragments derived from the piwi locus were used toprobe Northern blots of poly (A)+ RNA from wild-type male and femaleflies. Three non-overlapping transcripts, α (1.1 kb), β (0.64 kb), andc12 (3.1 kb) were identified in the region and only c12 was interruptedby piwi mutations. 9.36 kb of genomic DNA that encompasses the c12transcription unit was sequenced either using a Sequenase kit (U.S.Biochemical) or an ABI 377 Prism DNA Sequencer (Perkin-Elmer, AppliedBiosystems Inc). All sequence data was analyzed using the DNASTARsoftware package (DNASTAR, Madison, Wis.). Two additional transcripts,LD08487 (1.43 kb) and LD13681, were identified on the right side of theP-insertions by BLAST analysis (Altschul, et al., 1990) of genomicsequence.

[0284] Two overlapping cDNA clones which together represent the 3.1 kbfull length c12 mRNA and the LD08487 cDNA were sequenced. Comparisonbetween the genomic and the cDNA sequences precisely determined theintron-exon organization of these two transcription units. The LD13681transcription unit were also mapped by partial sequencing. Standardmolecular biology techniques were carried out according to Sambrook etal. (1989).

[0285] Transgenic rescue. The 6.8 kb HindIII/EcORV (pRc12; piwi) and 3.9kb PstI (pRLD; LD08487) genomic fragments were subcloned into the Pelement transformation vector pCaSpeR4 (Pirrotta, 1988). Transgenicflies were produced according to Spradling and Rubin (1982), using w;Δ2-3 Sb e/TM6 e (Robertson et al., 1988) embryos as recipients.Transgenes were then separated from the Δ2-3 transposase and introducedinto the homozygous piwi¹ background for fertility tests by geneticcrosses. Six independent pRc12 inserts and eight independent pRLDinserts were recovered. Rescue crosses were carried out at 25° C.

[0286] Northern blot analysis and piwi cDNA isolation. Poly (A)+ mRNAisolation, Northern blot preparation, and ³²P-labeled probe preparationby random-hexamer extension were according to Sambrook et al. (1989).For FIG. 2B, the probe was made from a 5.4 kb HindIII genomic fragmentencompassing the piwi transcription unit. The same probe was used toisolate a 2.1 kb partial cDNA from the pNB40 third instar library ofBrown and Kafatos, 1989. Using the sites within the polylinker (HindIIIat 5′ end and NotI at 3′ end), the 2.1 kb cDNA was subcloned intopBlueScript KS(+) (Stratagene), resulting in a construct denoted pDC1. Aprimer, pDC1 5′REV, 5′-ACGATAAGTTCTGTTAT-3′ (SEQ ID NO:11) was designeddownstream of the 5′ end of pDC1 and was used in combination withlibrary specific primers to screen the Tulle Hazelrigg ovarian cDNAlibrary (Friedman et al., 1990). A partially overlapping 500 bp PCRproduct was isolated, cloned into the pGEM-T vector (Promega, Madison,Wis.), and used to probe the same library. An overlapping 1 kb cDNAclone was isolated and subcloned into the SalI site of pDC1 to produce afull length 3.1 kb piwi cDNA construct denoted pDC2.

[0287] Characterization of piwi homologs from C. elegans and Homosapiens. The BLAST algorithm (Altschul et al., 1990) was used to searchthe non-redundant databases at the National Center for BiotechnologyInformation (NCBI) at the National Library of Medicine. The PrositeProtein Motif Database (DNAStar, Madison, Wis.) was also searched toidentify domains shared between piwi and other known proteins. The PIWIORF was used to query the SWISS-Prot database by BLAST for homologousproteins. Significantly, the search identified two C. elegans predictedproteins of unknown function. Expressed sequence tags (ESTs)corresponding to these two gene products were then identified as D2030.6(prg-1; GenBank accession No. Z73906) and C01G5.2 (prg-2; GenBankaccession NoU50068) from GenBank. Dr. Yuji Kohara (National Institute ofGenetics, Japan) kindly provided λZAPII phagemid clones corresponding toprg-1 and prg-2.

[0288] The piwi cDNA was further used to query the NCBI dbEST databaseand identified an EST, zw68h01.r1, isolated from a human testes cDNAlibrary, GenBank accession number M430311. This 0.9 kb EST clone wassequenced and used to screen an oligo-(dT) primed human testis cDNAlibrary (Clontech, Palo Alto, Calif.). A 2.3 kb cDNA clone correspondingto hiwi was isolated and sequenced.

[0289] Whole-mount tissue in situ hybridization. Ovarian whole-mounttissue in situ hybridization was performed as described in Yue andSpradling (1992) with DIG-labeled (Genius Kit, Boerhinger Mannheim).full length piwi cDNA from pDC2 as a probe. For detecting RNA in surfacecells, ovaries were digested 5 minutes with 50 μg/ml proteinase K. Underthis condition, only terminal filament cells and anterior sheath cells,but not any other surface cells, were stained with the piwi cDNA probe.The pBST control probe did not show any significant signal. Fordetecting RNA in inner germarial cells, the digestion was 7-10 minutes.Under this condition, the piwi probe revealed a hybridization pattern asshown in Results, the pBST control probe did not show any significantsignal, while the positive control oskar probe showed specific stainingin presumptive oocytes (Ephrussi et al., 1991). Staging of egg chambersis according to King (1970).

[0290] Genetic clonal analyses and germarial transplantation. The piwi¹and piwi² germline clones were generated with the FLP-DFS technique asdescribed in Chou and Perrimon (1996). y, w P[hsFLP]¹²;P[ovo^(D1)]^(2L), FRT^(40A)/CyO males were crossed to w; piwi¹,FRT^(40A)/CyO virgin females to produce y,w P[hsFLP]¹²;P[ovo^(D1)]^(2L), FRT^(40A)/piwi¹, FRT^(40A) progeny. Identical crosseswere performed with w; piwi², FRT^(40A)/CyO virgin females. The genotypeof the flies were verified both by Southern analysis for the presence ofpiwi P insertion and the FRT as well as by sterility tests bybackcrossing to the original piwi allele. Crosses were carried out fortwo days to produce transheterozygous progeny. After 2 days, adults weretransferred to fresh vials. Larvae were heat shocked on days 3 and 4 forone hour in a 37° C. water bath to induce mitotic recombination. Theheat-shocked females with germline clones were crossed to Ore-R malesfor fertility tests and their ovarian phenotype examined byimmunofluorescence microscopy.

[0291] To generate somatic clones of piwi¹ and piwi² the above describedpiwi¹, FRT^(40A)/CyO and piwi², FRT^(40A)/CyO strains were crossed to w;P[hsFLP]^(9F); P[w⁺; hs-NM], FRT^(40A)/CyO males as described in Xu andRubin (1993). Females of the genotype w, P[hsFLP]^(9F); P[w⁺; hs-NM],FRT^(40A)/piwi, FRT^(40A) were crossed to Ore-R males for fertilitytests. Clonal induction was carried out as described above except thatfemales of the appropriate genotype were subjected to an additional20-minute heat shock at 37° C. 90 minutes prior to dissection to inducethe expression of the myc tag. Germarial transplantation was carried outas described by Lin and Spradling (1993).

[0292] RNA-mediated interference (RNAi). Templates for in vitrotranscription were cloned into pBluescript KS(+) and transcribed usingeither T7 or T3 RNA polymerases according to the manufacturer's protocol(Megascript T7 and T3 kits, Ambion). prg-1 cDNA served as theexperimental RNA for injection while pBluescript KS(+) RNA was used as anegative control. RNA integrity was determined by gel electrophoresis;concentration was determined by a combination of UV spectrophotometryand ethidium bromide staining. Injections used an equimolar mixture ofuncapped sense and antisense RNA at a concentration of 10 mg/ml. Gonadalinjections of wild type N2 Bristol hermaphrodites was done as describedin Mello and Fire (1995).

[0293] After recovery and transfer to standard solid media, injectedanimals were transferred to fresh culture plates at 24 hours intervalsto facilitate the identification of phenotypic differences. Germlines ofprogeny produced between 6 and 48 hours after injection were examinedfor gamete differentiation either by DAPI staining or fertility. ForDAPI analysis, adult hermaphrodites displaying RNAi induced phenotypeswere fixed overnight in Carnoy's solution (60% ethanol; 30% acetic acid;10% chloroform; Sulston and Hodgkin, 1988). The mitotic index is definedas the ratio between the number of prophase, metaphase, anaphase, andtelophase nuclei and the total number of nuclei in the MPZ.

[0294] Piwi mutations eliminate the growth proliferation andself-renewing division of germline stem cells. piwi¹ mutant ovariescontain a normal number of mispositioned GSCs at the onset of oogenesisat the late third instar larval stage, which, however, leads to an equalor somewhat smaller number of gametes in the adult gonads that no longercontain GSCs (Lin and Spradling, 1997). This failure of germlinemaintenance could be due to: (1) the differentiation of GSCs withoutgrowth, proliferation and self-renewing divisions; (2) a defect in theasymmetry of GSC division, producing aberrant germ cells that eventuallydegenerate; and/or (3) a secondary defect influenced by abnormal ovarydifferentiation.

[0295] To examine whether the failure of germline maintenance is indeeda secondary defect due to abnormal ovary development, the ovarianmorphology of piwi² and piwi³ mutants which also fail to maintain GSCswas examined. The examination relied on Nomarski optics as well asmarkers that specifically identify germ cells, somatic cells, andspectrosomes/fusomes, germline specific organelles that indicateindividual stages of germ cell development (Lin et al., 1994; Lin andSpradling, 1995). These mutant ovaries show normal morphology at thethird instar larval stage. Their germline cells are normal in number andare correctly positioned along the medial plane of the ovary. Moreover,the expected number of terminal filaments are forming, so that at thepupal stage, the ovary differentiates normally, partitioning GSCs andtheir daughter cells correctly into individual germaria and ovarioles.GSCs are able to divide several times to provide a normal complement ofgerm cells to the germarium. Yet, GSCs subsequently fail to continueself-renewing divisions, and the existing germline cysts oftendegenerate during the, late pupal stage so that the adult ovariolescontain germlineless germaria and fewer egg chambers than expected.These observations indicate that the failure of GSC maintenance in piwimutants is not a secondary defect due to abnormal ovary development.

[0296] To examine whether the failure of GSC maintenance is due todifferentiation without self-renewing divisions or due to aberrantdivisions followed by degeneration, the ovarian development of the piwi¹mutant was examined more closely. The piwi¹ third instar larval ovariescontain a normal number of 50-70 presumed GSCs that are mis-positioned.However, these GSCs still seem to, and indeed must have, partitionedduring subsequent pupal development, since the number of ovariolesformed in most adult ovaries is within the normal range (13±3 in mutantvs. 15±3 in wildtype). Hence, the effect of piwi¹ on GSC mis-positioningis not essential for oogenesis.

[0297] The main oogenic defect of the piwi¹ mutant is thedifferentiation of GSCs without self-renewing divisions immediatelyfollowing the initiation of oogenesis. At this stage in wildtypeovaries, GSCs in 16-23 newly formed germaria have initiated asymmetricdivisions to generate multiple developing germline cysts. However, inmutant ovaries, GSCs and their immediate differentiated daughters,cystoblasts, are undetectable, as indicated by the absence ofspectrosome-containing germ cells. Instead, most ovaries containdifferentiated germline cysts whose number approximately equals that ofGSCs. These cysts are much larger in size and contain 2- to multi-cellstage fusomes, indicating their differentiating state. By the adultstage, most ovarioles contain only two normal or abnormal egg chambersderived from these cysts, but no other germline cells. This defectcontrasts the development of wildtype ovaries, in which ovariolescontain a fully developed germarium and a stage 1 egg chamber by the48-hour pupal stage, and have produced multiple egg chambers by adultstage. This observation indicates that the mutant GSCs havedifferentiated into germline cysts without self-renewing divisions. Theabnormal fusome morphology often seen in cysts suggest that they containmore or less than the normal number of 16 cells, which indeed was seenin adult egg chambers.

[0298] Thus, piwi is required for the self-renewing division of GSCsduring oogenesis in Drosophila. Confocal images showing third instarlarval ovaries from wild-type, piwi², and piwi¹ mutant flies,respectively, were prepared and stained with a germline specific markerVASA in green and spectrin in red to mark somatic cells andspectrosomes/fusomes. GSCs were positioned medially along theanterioposterior axis in the wild-type and piwi² ovary; however, GSCsare mispositioned in the piwi¹ mutant ovary. Spectrosomes (Sp) appear asred dots in germ cells. A 50 μm measurement bar was used in the analysisof the confocal images.

[0299] Confocal images of 24-hour pupal ovaries from wild-type and piwi¹mutant flies, respectively, were prepared, with wild type stained solelyfor VASA while piwi¹ mutant was stained for both VASA in green andspectrin in red. At this stage, the wild-type ovary has partitioned intoindividual germaria (Ge), which contain GSCs and early germline cysts.In piwi¹ mutant ovaries germ cells still appear to have beenpartitioned, although the partition is distorted by the highlydifferentiated large germline cysts containing elaborate fusomes (Fu).The number of cysts did not exceed the number of GSCs.

[0300] Confocal images of 48-hour pupal ovaries from wild-type, piwi²and piwi¹ mutant flies, respectively, were prepared and stained withVASA in green and spectrin in red. By this stage, in both wild-type andpiwi² mutant ovaries, germaria have fully developed and stage 1 eggchambers (S1) have budded off the germarium in synchrony due tocontinued GSC division. However, in piwi¹ mutant ovaries, the number ofgermline cysts remained unchanged, despite their continueddifferentiation.

[0301] A confocal image of a 72-hour pupal ovary from piwi² mutant flieswas prepared and stained with VASA in green and spectrin stained in red.At this stage, GSCs fail to self-renew and some existing germline cystsstart to degenerate. DAPI images of 0-1 day old adult ovarioles fromwild-type, piwi² and piwi¹ mutant flies, respectively, were alsoprepared. Both piwi¹ (L) and piwi² (H) mutant ovarioles typicallycontain two normal or abnormal egg chambers connected to a germlinelessgermaria (Ge). In contrast, wild-type ovarioles contain a long string ofdeveloping egg chambers produced continuously by the germarium. piwi³phenotype is indistinguishable from that of piwi².

[0302] Molecular cloning of Drosophila piwi. To study the molecularnature of the piwi-mediated mechanism, a 15 kb genomic region flankingthe piwi insertions was cloned. Candidate transcripts in the region wereidentified, and a 6.8 kb genomic fragment (pRc12) containing a 3.1 kbtranscript interrupted by piwi mutations (transcript c12) was able torescue the piwi phenotype completely (FIG. 1). An overlapping 3.9 kbgenomic fragment (PRLD) containing a 1.43 kb transcript immediatelyadjacent to the piwi mutations fails to rescue the piwi phenotype (FIG.1). Therefore, the c12 transcript is the piwi mRNA.

[0303]Drosophila PIWI represents a novel class of conserved proteins. Tostudy the structure of the piwi gene, two overlapping cDNA clones whichtogether represent the 3.1 kb full length c12 mRNA were isolated andsequenced. The c12 cDNA sequence contains a large open reading frame(ORF) starting at +84 bp and encoding a protein of 843 amino acidresidues with a predicted molecular weight of 97.2 kD (SEQ ID NOs:1 and2). Multiple stop codons in all three frames are present upstream of theputative start codon. Downstream of the ORF is a 432 bp 3′ untranslatedregion (UTR) followed by a 55-bp poly(A) tail not encoded in the genomicsequence. Thus, piwi RNA is present in piwi¹/CyO adult males andfemales. Northern blots including female and male lanes containingapproximately 10 and 1.5 mg of polyA⁺ RNA, respectively, were prepared.They were exposed for 3.5 and 12 hours, respectively, and a band wasobserved at 3.1 kb in each lane.

[0304] The PIWI protein is a highly basic (pl 9.6) novel protein with noobvious similarity to other known proteins or functional motifs in thedatabases. It is characterized by alternating basic and acidic regionsand is particularly basic over the C-terminal 100 amino acid residues.Hydropathy analysis indicates that the PIWI protein contains nosignificant local hydrophobic regions that could be potential signalpeptide or transmembrane domains. PSORT analysis (Nakai and Kanehisa,1992) predicts nuclear localization of the PIWI protein. The protein has21 conserved protein kinase C phosphorylation sites, 14 casein kinase 2phosphorylation sites, and 4 tyrosine phosphorylation sites, indicatingits potential as a phosphorylation target.

[0305] To determine whether the PIWI protein is conserved duringevolution, applicant searched for its homologous sequences at theprotein level and identified two ORFs of unknown function from C.elegans and an expressed sequence tag (EST, M430311) from a human testiscDNA library. Applicant isolated and sequenced cDNAs for the two C.elegans genes, herein named prg-1 and prg-2 (prg for piwi-related gene)to verify their homology to Drosophila piwi. The prg-1 and prg-2 genesshare 40.1% and 38.5% amino acid identity to piwi, respectively, overtheir entire length. In the C-terminal 104 amino acid region, thehomology increases to 55.8% and 56.7%, respectively. Moreover, prg-1 andprg-2 are 90% identical to each other over their full length and 98%identical at the C-terminus. This high degree of homology suggests thatprg-1 and prg-2 may represent a gene duplication event. The two clonesdiffer primarily in that prg-1 is 60 amino acids longer at theN-terminus than prg-2. Using ACeDB (Thierry-Mieg and Durbin, 1992),prg-1 was mapped to chromosome I between unc-15 and gld-1 in cosmidD2030 and prg-2 was mapped to chromosome IV, between unc-44 and smg-7 oncosmid CO1 G5.

[0306] To isolate human piwi homologs, the human EST (0.9 kb) clone wassequenced and used to screen a human testis cDNA library. A resulting2.3 kb partial cDNA, herein named hiwi(for human piwi), shows 47.1%identical amino acid sequence to the Drosophila piwi over its fulllength, with 58.7% identity at the C-terminus. No piwi-related sequenceswere found from bacteria or yeast genomes whose entire sequences areknown. This is consistent with the stem cell-related function of piwiand indicative of piwi family-like genes specific for multicellularorganisms.

[0307] In addition to the above piwi family homologs, a large number ofputative and known proteins were identified from various animals andplants which share significant homology with piwi solely at theirrespective C-termini. This indicates that the piwi family is a largenovel gene family. Within this family are 13 additional putative C.elegans proteins and three A. thaliana proteins, two of which arerequired for meristem cell divisions (Moussian, et al., 1998; Bohmert,et al., 1998). Particularly, between piwi and the three Arabidopsisgenes, ZWILLE (ZLL) argonaute (ago), and argonaute-like (Moussian etal., 1998; Bohmert et al., 1998), an overall homology of approximately20% amino acid identity was observed. The homology increases to 32% to52% identity in four regions, 30-100 amino acid residues each, locatedthroughout the length of the PIWI protein, including the highlyconserved C-terminal region. Given the role of ZLL and ago in meristemcell division (Moussian et al., 1998; Bohmert et al., 1998), thishomology may have important implications for a conserved stem cellmechanism.

[0308] PIWI, PRG-1, PRG-2, and HIWI differ from ZLL and AGO proteins,and especially from the 13 additional putative C. elegans proteins,predominantly at the N-terminus, suggesting that this region may beinvolved in piwi-specific function. The C-terminal conservation suggeststhat this region of PIWI may contain a novel functional domain thatplays an important role for the general activity of these proteins indiverse biochemical processes, with the N-terminus rendering thespecificity of the activity.

[0309] To examine the C-terminal region of homology more closely, thesequences were aligned using Block Maker™, which reveals characteristicregions of protein families (Henikoff et al., 1995) (FIG. 2). BlockMaker™analysis identified a 43 amino acid domain conserved among all 22proteins, within which five residues are absolutely conserved withdefined spacing. Eight more residues are also conserved with definedspacing among all known genes across the phyla except for several C.elegans ORFs with unknown function. This region is referred to herein asthe PIWI box and represents a novel conserved functional motif. PIWI,that is the piwi family of gene products, thus represents a novel classof evolutionarily conserved proteins with conserved functionality, asdescribed herein below.

[0310]Drosophila piwi mRNA is present in both the germline and somaduring oogenesis. To investigate in which cells piwi is expressed toregulate GSC division, the expression pattern of piwi during oogenesiswas examined by in situ hybridization of whole mount Drosophila ovarieswith DIG-labeled DNA probes prepared from the piwi cDNA clone. piwi mRNAis detected specifically in the somatic terminal filament cells apicalto GSCs in the germarium and anterior sheath cells as well as in thegermline. In the germline, it is first abundantly expressed in region 2of the germarium where 16-cell germline cysts are formed, persists at alower level through stages 1-6 of oogenesis, is at its lowest levelbetween stage 7-9, becomes strongly expressed again at stage 10, andeventually accumulates in early embryos (for staging, see King, 1970).Given that removing sheath cells does not affect oogenesis whereas theterminal filament cells play a role in regulating GSC division (Lin andSpradling, 1993), it is likely that the somatic expression in theterminal filament is responsible for piwi function in regulating GSCdivision.

[0311] Thus, piwi mRNA expression in Drosophila ovaries wascharacterized. A complete Ore-R ovariole was studied and showed germlinepiwi expression in region II of the germarium (Gell) and in early stageegg chambers up to stage 10 (S10) was studied. In the germarium, thepiwi RNA was also detected in the terminal filament cells (TF) andepithelial sheath cells (ES) apical to GSCs. piwi RNA was also observedto be uniformly present in early embryos. The aforementioned structureswere reviewed on slides, and the slides included bars denoting 50 μm, 10μm and 50 μm for ovariole, germarium and early embryos, respectively, tofacilitate review.

[0312] Piwi expression in apical somatic cells regulates GSC divisionwhile its expression in the germline Provides a maternal component forembryogenesis. To examine the roles of somatic and germline piwiexpression, genetic clonal analyses on piwi¹ and piwi² mutations wasconducted. First, the germline requirement of piwi was tested bygermline clonal analysis using the FLP-DFS technique (Chou and Perrimon,1996). The ovo^(D1) dominant mutation blocks oogenesis at the verybeginning stage in a cell-autonomous manner, so that piwi⁺ ovo^(D1)females contain germ cells that never differentiate beyond thegermarium. In this ovo^(D1) background, applicant generated germlinecells homozygous for either the piwi¹ or piwi² mutation by applying aheatshock treatment at the second and early third instar larval stage.The resulting adult females developed some completely normal ovariolesin which germline cells at all stages of oogenesis were observed, andmature eggs were continuously being produced (see Table 3).

[0313] This result demonstrates that removing the piwi⁺ ovo^(D1)chromosome from the germline allows oogenesis, including GSC division,to occur normally. Thus, the requirement of piwi for GSC division doesnot reside in the germline but in somatic cells.

[0314] Eggs produced from homozygous piwi¹ or piwi² GSC clones arearrested in embryogenesis, not rescuable by the paternal piwi⁺ gene (seeTable 3). Approximately 30% of the arrested embryos show severe mitoticdefects during cleavage stage. The remaining embryos show variousmorphological defects during late embryogenesis, including a highfrequency of severe deformation of the head region. These defectsdemonstrate that piwi expression in the germline provides an essentialmaternal contribution for embryogenesis.

[0315] To determine whether piwi is required in somatic cells outsidethe ovary for GSC maintenance, wildtype germaria were transplanted intothe abdominal cavity of homozygous piwi² and wildtype females, whichproduced 17±0 egg chambers (n=13) after 7 days of incubation in thehost. Parallel transplantation into wildtype host yielded 16±2 eggchambers (n=5), indicating that the wildtype germaria continuedoogenesis at a normal rate in the piwi² females. Hence, piwi is notrequired in extra-ovarian cells for GSC maintenance.

[0316] To determine in which group of ovarian somatic cells piwi isrequired for GSC maintenance, applicant used a similar FRT-mediatedtechnique for somatic clonal analysis of piwi¹ and piwi², marking thepiwi⁺ chromosome with a transgenic myc tag so that homozygous piwi⁻somatic cells are identified by their lack of myc epitope expression (Xuand Rubin, 1993). By inducing mitotic recombination at the second andthird instar larval stages, piwi somatic clones were generatedthroughout ovarioles, with some egg chambers completely covered by piwimutant follicle cells. These egg chambers develop normally, indicatingthat piwi function is not required in follicle cells for egg chamberdevelopment. Since follicle cells are derived from their precursor cellsin region II of the germarium (Margolis and Spradling, 1995), thissuggests that piwi is not required in somatic cells from germarialregion II on for GSC division and ovarian development. These results,together with the piwi expression pattern in situ, suggest that piwi isrequired in somatic cells in the anterior-most tip of the germarium toregulate GSC division.

[0317] Thus, piwi function is dispensable in the germline anddifferentiated follicle cells for GSC division. A pair of ovariescontaining ovarioles with homozygous piwi⁻ germline clones and ovarioleswith piwi⁺ ovo^(D1) germline stained for DAPI to mark DNA and for VASAto mark germ cells for observation via confocal microscopy. The piwi⁺ovo^(D1) germline cells were arrested at the beginning of oogenesis inthe germarium due to the ovo^(D1) mutation. By contrast, in ovarioles inthe left ovary containing piwi⁻ germline clones, GSCs divided normally,giving rise to a progression of morphologically wild-type egg chamberswhich eventually develop into mature eggs. This indicates that piwi isnot required in the germline for GSC division and subsequent steps ofoogenesis. To facilitate confocal microscopy, bars denoting 100 μm wereemployed in each slide.

[0318] Confocal microscopy was also employed to, show that piwi⁻ clonesin differentiated follicle cells do not effect normal GSC division andegg chamber development. Egg chambers were stained with DAPI and amonoclonal anti-MYC antibody. Absence of MYC staining was indicative ofhomozygous piwi-clones. Induction of somatic clones around an entireearly stage egg chamber had no effect on GSC division and oogenesis.Similarly, removing piwi function from some follicle cells of laterstage egg chambers also had no, effect on oogenesis. Therefore, piwifunction is dispensable in differentiated follicle cells for oogenesisincluding GSC division. To facilitate confocal microscopy, bars denoting50 μm were employed in each slide.

[0319] piwi-Like Genes in C. Elegans are Also Required for GermlineSelf-Renewal. The high degree of sequence homology between piwi and itshomologs in other organisms suggests a potential functionalconservation. This hypothesis was tested in C. elegans. Guo and Kemphues(1995) have shown that the injection of specific antisense RNA into thegermline syncytium of C. elegans eliminates maternal and zygotic geneactivity, producing a gene-specific loss-of-function effect that maypersist through several generations. This technique, as refined by Fireet al. (1998) and termed RNA-mediated interference (RNAi), was used toassess the function of prg-1 and prg-2. Given the extremely highhomology between prg-1 and prg-2, an anti-prg-1 RNA was used forinjection to interfere with the function of both genes. The F1 progenyof the injected worms were designated as prg-RNAi worms for simplicity.

[0320] In wild-type C. elegans, two germline precursor cells, Z2 and Z3,give rise to approximately 2000 germ cells in the adult hermaphrodite.Germline proliferation occurs throughout most of larval development(L1-L4) and continues in the adult. This proliferation and maintenanceof the germline requires signals from the DTC at the tip of each gonadalarm. During gonadal development, DTC migration results in the formationof two U-shaped gonadal arms by the L4 stage. Germline proliferation islimited to the distal end of each arm, forming a mitotic proliferationzone (MPZ) which serves as the GSC equivalent in C. elegans. Movingproximally, near the U turn of the gonad, germ cells enter meioticprophase and then further differentiate into gametes at the proximalhalf of each arm, producing sperm in L4 and then oocytes in youngadults.

[0321] The phenotype of prg-RNAi worms was examined both by quantitatingtheir fertility and by assessing their gonadal and germline developmentvia DAPI staining and Nomarski optics. As controls for RNA injection,worms were injected with BlueScript plasmid RNA sequences.

[0322] The prg-RNAi worms show three major aspects of germline defects.First, as indicated by the distribution of the number of progeny per F1animal (FIG. 3), the fertility difference between the prg-RNAi andcontrol worms is highly significant (t value=7.93, p<0.0001). Onaverage, prg-RNAi worms produced 92±53 progeny (n=99), with 75.5% ofprg-RNAi worms giving rise to fewer than 125 progeny. By contrast,control RNAi animals on average produced 191±40 progeny (n=20), with 90%of these animals giving rise to greater than 125 progeny. Thesignificant reduction of the fertility, as well as other defectsdescribed below, may still only reflect a partial loss of prg-1 andprg-2 function, since the RNAi technique is known to phenocopy partialrather than complete loss-of-function mutants (Kuwabara, 1996).

[0323] Second, DAPI staining reveals a dramatic shortening of MPZ inprg-RNAi worms. On average, adult prg-RNAi worms exhibited a 50%reduction in the number of mitotic nuclei as compared to controlanimals. Associated with the reduction of the mitotic zone is a gonadalshortening. 0.57% of prg-RNAi worms (n=287) exhibited a moderate tosevere shortening. In the most severe case, the gonadal arm never madethe U-turn. The number of sperm produced in these worms is greatlyreduced as well. By contrast, only 7% of the BlueScript-RNA-injectedcontrol animals (n=548) exhibited a mild gonadal shortening. The aboveobserved gonadal defects in prg-RNAi worms indicate that prg-1 and prg-2are essential for germline proliferation and maintenance. Since thegonadal shortening may be due to a defect in DTC migration, it is alsopossible that prg-1- and prg-2 play a role in proper gonadogenesis.

[0324] Thus, reducing prg activity results in reductions in both the MPZand the mitotic index. Variable defects in both the size of the MPZ andthe mitotic index were observed by DAPI staining and preparation ofphotomicrographs of same. The MPZ in each micrograph was denoted inwhite brackets. In moderate to severely defective animals, a 50%reduction in the number of mitotic nuclei and a concomitant five-foldreduction in the mitotic index were typically observed. A measurementbar was included in the wild type photomicrograph and denoted 100 μm forall panels.

[0325] In addition to the MPZ shortening, the mitotic index in theremaining mitotic zone is further reduced by 5.5 fold from (7.1±2.2)%(n=1,339 nuclei, n_(s)=10 worms) in the control RNAi injected worms to(1.3+0.8)% (n=1,409 nuclei, n_(s)=10 worms) in prg-RNAi worms withmildly to moderately shortened gonads. This indicates the important roleof prg-1 and prg-2 in maintaining the mitotic ability of the germlinestem cell nuclei.

[0326]Drosophila piwi is required for the self-renewing divisions ofstem cells in the germline. The above experimentation demonstrates thatpiwi provides for female and male GSC maintenance predominantly bypromoting the self-renewing division of GSCs. In a germarium, itnormally takes at least 10 hours for a GSC division to occur and. 5 daysfor a cystoblast to develop into a stage one egg chamber (King, 1970;Lin and Spradling, 1993). Necrosis and apoptosis during oogenesis arealso easily detectable. Thus, examination of the piwi mutant phenotypeat 24-hour intervals immediately prior to the onset of oogenesis issufficient to monitor every major event in germline proliferation,differentiation, or cell death. Under these conditions, GSC divisionswould have been detected as an increased number of germline cysts,whereas cell death process would have been easily detected by lookingfor pycnotic nuclei and necrotic bodies via DAPI staining and Nomarskioptics (Lin and Spradling, 1993; Forbes et al., 1996a, b; Lin. andSpradling, 1997). Numerous cases of GSC divisions and cell death in thegermline during Drosophila oogenesis have been detected using thesecriteria (Lin and Spradling, 1993; Forbes et al., 1996a, b; Lin andSpradling, 1997).

[0327] Despite their high sensitivity, the techniques followed in thisExample, even though more systematic, did not detect either GSC divisionor cell death in piwi mutants. Instead, immediately following the onsetof oogenesis, only differentiating cysts were detected in piwi¹ mutantovaries, with the cyst number generally corresponding to the number ofGSCs. Although this does not rule out the possibility that infrequentGSC divisions or cell death might have been missed, which might accountfor the smaller-than-expected number of gametes sometimes observed inadult piwi mutants, these factors do not weaken the conclusion that thepredominant defect of piwi mutants is the differentiation of GSCswithout accompanying self-renewing division.

[0328] A number of elegant studies have identified genes that regulategermline proliferation. In mammalian systems, mechanisms mediated byparacrine factors such as the Steel factor (the ligand of the c-Kitreceptor tyrosine kinase), leukemia inhibitory factor (LIF), and basicfibroblast growth factor (bFGF), control the proliferation and survivalof primordial germ cells, i.e., the precursors of GSCs (reviewed in Lin,1997). In C. elegans, the LAG-2/GLP-1 pathway is essential for theself-renewal of a stem-cell equivalent population of germline nuclei inthe gonad (reviewed in Kimble and Simpson, 1997). In the Drosophilaovary and testis, the BAM-mediated mechanism is involved in the furtherdevelopment of the differentiated GSC daughter (McKearin and Ohlstein,1995); the dpp pathway plays a similar role in testis (Matunis et al.1997). More recently, this pathway has been shown to be essential forGSC division and maintenance in the ovary (Xie and Spradling, 1998).Among these discoveries, piwi is the first gene known to mediate asomatic signaling mechanism to regulate the division and maintenance ofGSCs in Drosophila. It therefore provides an important entry point forstudying the somatic control of stem cell division in the germline.

[0329] The piwi gene family and the conservation of a stem cellself-renewing mechanism. The identification of prg-1 and prg-2 in C.elegans and hiwi in human suggest that piwi represents a novel class ofevolutionarily conserved genes (described herein as the “piwi family”)with potentially important functions in GSC self-renewal. Among thisclass of genes, the significantly higher homology between piwi and hiwias compared to that between piwi and prg-1/prg-2 suggests that hiwifunction is closer to piwi. Consistent with this, GSC division andgametogenesis in humans are much more similar to that in Drosophila thanthat in C. elegans whose gonads contain syncytial mitotic germlinenuclei which divide symmetrically and are capable of self-renewing onlyas a population. The RNAi experiments described herein, which likelyinterfered with the activity of both prg-1 and prg-2 due to theirextremely high homology, caused germline depletion similar to that inpiwi mutants. This suggests that the piwi-mediated mechanism in germlineself-renewal is conserved even in this evolutionarily distant organismwithout stereotypic GSCs.

[0330] The conservation of the piwi-mediated mechanism appears to extendto the plant kingdom as well. A level of homology between PIWI and ZLLand AGO was observed. Intriguingly, ZLL is essential for maintainingstem cells of the shoot meristem in an undifferentiated state during thetransition from embryo-specific development to repetitive organformation through the self− perpetuating shoot meristem divisions(Moussian et al., 1998). AGO also plays an important role in maintainingnormal apical shoot meristem function (Bohmert et al., 1998). Thus, thehomology between piwi and ZLL and ago further suggests the existence ofa family of novel genes essential for stem cell division in diverseorganisms.

[0331] The conserved piwi mechanism may mediate cell-cell interactions.In Drosophila, genetic mosaic and piwi expression analyses togethersuggest that piwi function is required in the apical non-mitotic somaticcells to control GSC division. Similarly, in A. thaliana, it is thoughtthat ZLL is required to maintain the undifferentiated state of shootmeristem stem cells by relaying positional information, possibly bymediating cell-cell interactions within the center of the shoot meristem(Moussian et al., 1998).

[0332] Stem cells are characterized by two common properties that extendacross diverse species: the capacity for self-renewal and the ability togive rise to numerous progeny that are fated for further differentiation(for review see Lin, 1997; Morrison et al., 1997). Although significantprogress has been made in identifying genes important for stem cellfunction, no common molecular mechanism shared by diverse stem celltypes in diverse organisms has been characterized with respect to thosetwo basic stem cell properties. The piwi gene family represents thefirst class of such genes. The analysis of the piwi gene family,therefore, provides an important first step towards the elucidation ofmolecular mechanisms underlying stem cell divisions.

[0333] The analyses of this Example were extended to homologs in C.elegans, an isolated human homolog (hiwi), and identified two piwi-likegenes in Arabidopsis which are known to maintain meristem cells. Thiswork reveals a new class of genes that play an essential role in stemcell division in multicellular organisms ranging from invertebrates tohumans and plants. TABLE 2 piwi¹ rescue results by pRc12 and pRLD^(a,b)pRc12; pRLD; piwi¹/piwi¹ piwi¹/CyO piwi¹/piwi¹ piwi¹/piwi¹ females 0(136)  100 (45)  100 (39) 0 (81) males 0 (119) 97.8 (47) 88.4 (43) 0(64)

[0334] TABLE 3 piwi function in GSC division is soma-dependent, whileits function in embryogenesis is germline dependent* FLP/FRTovo^(D1)-induced Heatshock N Avg. # eggs germline clones % embryoslaid/female hatched (a) piwi¹, FRT/ovo^(D1), FRT × Oregon^(R) males + 3750.4 0 (b) piwi², FRT/ovo^(D1), FRT × Oregon^(R) males + 26 66.7 0 (c)piwi¹, FRT/ovo^(D1), FRT × Oregon^(R) males − 44 0 na (d) piwi²,FRT/ovo^(D1), FRT × Oregon^(R) males − 26 0 na (e) ovo^(D1), FRT/CyO ×Oregon^(R) males + 20 0 na (f) ovo^(D1), FRT/CyO × Oregon^(R) males − 100 na # normal GSC division. However, none of the eggs develop beyondembryogenesis, indicating the germline piwi expression is required as amaternal component for embryogenesis. The ovo^(D1), FRT control femaleslay no eggs either with or without heatshock.

Example 2 Molecular Cloning and Expression Analysis of Hiwi, A HumanHomolog of the Drosophila Piwi Gene Essential for Germline Stem CellDivision and Embryogenesis

[0335] piwi is a novel gene first identified in Drosophila and wasdemonstrated to be essential for germline stem cell development inExample 1. In this Example the cloning of a 3.47 kb human piwi homologthat includes the whole open reading frame through cDNA libraryscreening and 0.5° RACE is described. Sequence analysis showed highidentity of HIWI to Drosophila PIWI, especially in the C-terminal domainof the protein. Chromosome mapping showed located hiwi to Chromosome 12q24. 33, approximately 130 kb from the AFM 295 ye5 marker. Northern blotanalysis revealed that hiwi is only expressed in the testis among humanadult tissues. The sequence and expression homology between human andDrosophila piwi genes suggested that piwi is conserved during evolutionand plays an important role during early stages of spermatogenesis inhuman.

[0336] Human spermatogenesis followed a precise and coordinated mannerin the seminiferous epithelium of testis (reviewed in Bellve, 1979;Thomas et al., 1989; Hoog, 1995). During this period the germ cellsundergo a series of stage-specific morphological transitions, beginningwith the proliferation and renewal of spermatogonia, followed by meiosisof spermatocytes, and finished by differentiation of spermatids duringspermiogenesis (Oakberg, 1956a; Nebel et al., 1961; Belive et al., 1977;Russel et al., 1990). Closing to the basal membrane, type Aspermatogonia are stem cells (Bartmanska and Clermont, 1983; DeRooij,1988), undergoing a series of mitoses to give rise to more type Acells or produce type B spermatogonia which passes through a mitoticdivision to differentiate into spermatocytes.

[0337] Spermatocytes need to undergo a final round of DNA replicationbefore entering meiosis. During meiosis, the synaptonemal complex andthe recombination nodule become associated with chromosomes, which wasthought to be important for two basic tasks in meiosis, the reduction ofploidy level and generation of new combinations of genes (reviewed invon Wettstein et al., 1984). The most radical morphological changesoccurred during spermiogenesis (Oakberg, 1956b). Based on acrosomeformation and nuclear condensation, the round spermid differentiatedinto'mature spermatozoa which released into the lumen indicating the endof the spermatogenesis (reviewed in Bellve and O'Brien, 1983).

[0338] The proliferation and differentiation of germ cells in theseminiferous tubule has been demonstrated to be associated with adjacentsomatic cells, the Sertoli cells (reviewed in Russel and Griswold, 1993;Griswold, 1995; Bitgood et al., 1996; de Miguel et al., 1997). Thetubule of human testis is composed of germ cells in all stages ofspermatogenesis. From basal membrane to lumen are cells ranging fromstem cell to differentiated cell state and from proliferation todifferentiation stage. Particular cell at particular developmental stageis associated with others at given sites in the tubule, the least maturestem cells are located furthest outside, while the most maturespermatozoa are located inside the seminiferous epithelium. The specifictubule organization and cellular association thus provide a good modelsystem to study germ cell development.

[0339] Genes expressed in male germ cells had been identified in highmammals (Kramer and Erickson, 1982; Gold et al., 1983; Thomas et al.,1989; Wolgemuth and Watrin, 1991; Hoog, 1995). Some of these genes wereexpressed exclusively during spermatogenesis (reviewed in Willison andAshworth, 1987; Erickson, 1990; Sandlow et al., 1997; van Roijen et al.,1998), while others were expressed only in the later. stages ofspermatogenesis (Shankar et al., 1998; Zhu and Naz,1998). However, veryfew genes had been demonstrated that was expressed specifically in theearly stage of spermatogenesis, in the stem cells (Menke et al., 1997).

[0340] To study the mechanisms of piwi family function in germline stemcells in various organisms during evolution and especially its role inhuman beings, molecular cloning and expression analysis of human piwifamily homolog was performed.

[0341] 5′-RACE. A 2.3 kb partial human piwi family cDNA clone was firstisolated from adult human testis cDNA library through library screening.To obtain the 5′ end of this gene, a cDNA synthesis primer and a genespecific primer has been designed. Adult human testis Poly A+ RNA waspurchased from Clontech, Palo Alto, Calif. First-strand cDNA wasobtained by using the above cDNA synthesis primer and AMV reversetranscriptase (Clontech, Palo Alto, Calif.). Second-strand synthesiscDNA synthesis was performed with second-strand enzymes bought fromClontech, Palo Alto, Calif. subsequently. Using the above gene specificprimer and an adaptor primer obtained from Clontech, Palo Alto, Calif.,an 1.4 kb fragment has been obtained through 5′-RACE PCR by usingMarathon™ cDNA Amplification Kit (Clontech, Palo Alto, Calif.) and hasbeen cloned into a pGEM-T Easy vector (Promega, Madison, Wis.)subsequently.

[0342] Construction of a full length human piwi homolog and its sequenceanalysis. The fully-length hiwi cDNA was generated in a modified pGEM-TEasy vector (Promega, Madison, Wis.) by partial digestion of above twooverlapping cDNA fragments with SacI and subsequent ligation with T₄ DNAligase (Gibco). The full length human piwi family homolog was sequencedfully on both strands using an ABI 377 Prism™ DNA Sequencer(Perkin-Elmer, Applied Biosystems) and analyzed using the DNAStar™sequence analysis' package (DNASTAR, Madison, Wis.). Homologouscomparison between hiwi and its homologs was carried out using theMegalign™ program from the DNAStar™ package.

[0343] Chromosome mapping. Radiation Hybrid mapping against Stanford G3Human Hamster panel was used for chromosome mapping for hiwi.

[0344] Northern Blot Analysis. A 0.9 kb EST fragment used for Northernblot analysis was purchased from ATCC (American Type Culture Collection)and was subcloned into a pBlueScript KS(+) vector (Stratagene) withEcoRI and HindIII. The 3.47 kb plasmid DNA obtained from 5′RACE was usedto make sense and antisense RNA probes for in situ hybridization. Theabove plasmid DNA were linearized with appropriate restriction enzymes.Using these linearized template DNA, sense and antisense RNA probes forNorthern blot analysis and in situ hybridization were transcribed fromthe T7 and SP6 promoters with appropriate T7 and SP6 RNA polymerase(Gibco), following the protocols suggested by manufacturers. Probes usedfor Northern blot analysis were labeled by ³²P-UTP (Amersham).

[0345] Premade Northern blot of high-quality poly A+ RNA from differenthuman tissues (Human Multiple Tissue Nothern Blot II) was purchased fromClontech, Palo Alto, Calif. Human β-actin cDNA probe (Clontech, PaloAlto, Calif.) was used as a positive control. ExpressHyb™ HybridizationSolution (Clontech, Palo Alto, Calif.) was used as hybridization bufferfor Northern analysis. The hybridization for both sample blot andpositive control was performed under 68° C. for 2 hours followed by 2washes in 2×SSC, 0.05% SDS for 50 minutes at room temperature, andanother 2 washes in 0.1×SSC, 0.1% SDS for 40 minutes at 50° C. Thesample blot was exposed at −80° C. for 10 hours while the positivecontrol was exposed for 2 hours at the same temperature.

[0346] Cloning of full-length human piwi cDNA clone. An isolated 2.3 kbpartial human piwi cDNA clone from a human testis library by libraryscreening is described in Example 1. To obtain the 5′ end of this gene,by using a gene-specific primer, a 1.4 kb cDNA fragment containingoverlapping sequence was obtained through 5′RACE and subcloned. Toobtain a full length human piwi family clone, the above overlapping cDNAclones were partially digested. Fragments of the correct sizes weresubsequently ligated and subcloned into a pGEM-T Easy vector (FIG. 4) togenerate the 3472 bp full-length human piwi family (hiwi) that includesan open reading frame (ORF) encoding a protein of 861 amino acids, witha predicted molecular weight of 94.71 kDa (FIG. 7 and FIG. 8). The ORFstarts at 167 bp and stops at 2749 bp. Upstream of the start codon is a166 bp 5′untranslated region (5′UTR). Downstream of the stop codon is a723 bp 3′untranslated region (3′UTR).

[0347] Protein sequence analysis showed that HIWI is 36.6% identical toDrosophila PIWI at amino acid level over its full length and 58.7%identical at the C terminal 104 amino acid residues. HIWI also shares33.4% and 33.1% identity with two C. elegans PIWI family homologs PRG-1and PRG-2 over its full length while the percentage increased to 57.7%and 58.7% at the C-terminals, suggesting piwi family genes and geneproducts are conserved during evolution. In addition, the higher degreeof sequence homology between the C terminals suggests a conserved roleof the C-terminal domain of PIWI in protein function.

[0348] Chromosomal mappinq of hiwi. By using Radiation Hybrid mappingmethod against the Stanford G3 panel, human piwi was mapped tochromosome 12q 24.33, approximately 130 kb from the AFM 295 ye5 marker.

[0349] Tissue distribution of hiwi in adult human. Northern blotanalysis were performed to study the tissue distribution of hiwi inadult human tissues. A variety of human tissues were examined, includingspleen, thymus, prostate, testis, ovary, small intestine, colon andperipheral blood leukocyte. Among them, two transcripts were detected intestis: a more abundant 3.6 kb band and a less abundant band of 8.1 kb.No expression was found in other analyzed tissues, consistent with theknown functions of hiwi in GSC division.

[0350] An autoradiograph of Northern blot analysis of hiwi mRNA in adulthuman and mouse tissues was thus prepared. The expression of hiwi mRNAin different tissues: spleen; thymus; prostate; testis; ovary; smallintestine; colon; peripheral blood leukocyte. Two hiwi transcripts of3.6 kb and 8.1 kb are visible in testis. Human β-actin cDNA probe wasused as a positive control for the Northern blot with above tissues.

[0351] The specific expression of hiwi in the testis is contemplated tomodulate the mechanism of some human genetic diseases related to testismalignancy and/or infertility. hiwi and other mammalian piwi familygenes play an important role in germline stem cell division and otherstem cell-related processes and are thus contemplated to be useful inthe therapeutic methods, the screening methods, and the diagnostic andprognostic methods of the present invention set forth herein.

Example 3 miwi, a Murine Homolog of piwi, Encodes a Cytoplamsic ProteinEssential for Spermatogenesis

[0352] This Example shows cloning, expression analysis, and functionalanalysis of a mammalian member of the family, miwi, that is essentialfor spermatogenesis. miwi shares significant homology with other piwifamily members, especially in the C-terminal PIWI box region. It encodesa cytoplasmic protein that is specifically expressed in spermatocytes ofthe adult testis. Consistent with its expression pattern, miwi-null miceare viable and female fertile, but completely male sterile. Histologicaland molecular marker analyses suggest that spermatogonialdifferentiation appears to be delayed, apoptosis increases drasticallyin spermatogenic cells at all stages, and spermatogenesis is arrested bythe round spermatid stage, blocking spermiogenesis. These analysesreveal novel functions of a piwi family gene in mammalianspermatogenesis. Moreover, deletion of the C-terminal PIWI box region ofMIWI leads to spermatogenic defects indistinguishable from the completeMIWI deletion, suggesting that this highly conserved region is essentialfor the function of PIWI family proteins.

Materials and Methods

[0353] miwi cloning and Northern analysis. Multiple cDNAs representing3.5 kb of the 3′ end of miwi mRNA were isolated from mouse testis cDNAlibraries obtained from Clontech Laboratories, Inc. (PalQ Alto, Calif.)and from Dr. Quangquan Zhao using a human EST clone zw68h01.r, a hiwicDNA, (Cox et al., 1998) as an initial probe. To retrieve cDNA sequenceof the entire ORF, 5′RACE was performed using the MARATHON™ cDNAAmplification Kit (Clontech Laboratories, Inc. of Palo Alto, Calif.)with 5′-TGCACTGCCAGGTCCTTCATCAC-3′ (SEQ ID NO:12) as the reversetranscription primer and 5′-GGCCAGTCATTTTCCAGtCAGCTCAGGTG-3′ (SEQ IDNO:13) as the miwi-specific PCR primer. Together, these miwi cDNAs cover4.06 kb miwi mRNA sequence, including the complete ORF and 3′-UTR. Sincethe 4.7 kb miwi transcript is at least 20-fold more abundant than the6.5 kb transcript, these cDNAs likely represent the 4.7 kb transcript.To obtain a miwi genomic sequence, a miwi probe was used to isolateclones comprising 42 kb of genomic DNA from a lambda library provided byDr. J. Rossant via Dr. J. Klingensmith. 29.2 kb of genomic DNA wassubcloned into the pBlueScript KS (+) vector (Stratagene of La Jolla,Calif.) and sequenced. The alignment of this genomic region to miwi cDNAreveals, the intron/exon structures. Northern analysis was conductedusing a 2.0 kb miwi cDNA as a probe and blots purchased from Clontech(Palo Alto, Calif.) (2 μg poly(A)+ RNA/lane).

[0354] Mapping the miwi locus. The miwi locus was located by PCR-basedmeiotic mapping against the European Collaborative InterspecificBackcross (EUCIB) panel. A primer pair from the 3′ UTR region of themiwi cDNA was used to amplify the corresponding region in 50 EUCIBbackcrosses. The PCR products were heat-denatured and separated bynondenaturing gel electrophoresis to detect motility shift caused bysingle stranded conformation polymorphism. Non-polymorphic andpolymorphic samples were scored as 1 and 2, respectively. The scoreswere then submitted to the EUCIB on-line database to obtain the mappingdata.

[0355] Antibody generation. Two miwi cDNA fragments corresponding toamino acid residues 239-524 (MIWI34) and 532-862 (MIWI56) were clonedinto the pGEM®-EX vector (Promega of Madison, Wis.) for bacterialoverexpression of the peptides. Inclusion bodies containing theoverexpressed peptides (>80% purity) were purified from the bacteria anddissolved in solubilization buffer (7M urea). The solubilized inclusionbodies were used to immunize rabbits, performed by Cocalico Biologicals,Inc. (Reamstown, Pa.). Antisera R132 and R133 were generated usingMIWI34 as an antigen, while antisera R134 and R135 were generated usingMIWI56 as an antigen. Antibodies were affinity purified byimmuno-blotting (Harlow and Lane, 1988). All of the four antibodiesrecognize the same protein on western blots. Antisera R133 was used forall the experiments described herein, and is referred to as “anti-MIWIantibody”.

[0356] RNA in situ hybridization. Digoxygenin-rUTP-labeled sense andantisense RNAs were translated from a 1.9 kb miwi cDNA fragmentcorresponding to amino acids 137-767 and subsequently used as probes forin situ hybridization on 8 μm testis cryosections. Hybridization wascarried out at 60° C. overnight in a buffer (pH6.5) containing 50%formamide, 1.3×SSC, 5 mM EDTA, 0.5% CHAPS, 0.2% TWEEN-20™, 50 μg/mlyeast RNA, 100 μg/ml heparin, and 0.1-1 μg/ml sense or antisense RNAprobe. After post-hybridization washes, sections were probed withalkaline phosphatase-conjugated anti-dioxigenin antibody (1:1000, RocheMolecular Biochemicals of Basel, Switzerland) at room temperature for 1hour. The enzymatic reaction was carried out using FAST RED™ compound(Roche Molecular Biochemicals of Basel, Switzerland) as a substrateaccording to the manufacturer's instruction.

[0357] Western blotting and immuno-microscopy. For Western blotting,dissected mouse tissues were homogenized in 3-5 volumes of sample buffer(5% 2-mercaptoethanol, 3% SDS, 10% glycerol, 62.5 mM Tris-HCl pH6.8),boiled for 5 minutes, and separated by 10% SDS-PAGE (501g/lane).Proteins were transferred to a GENESCREEN™ filter (Genetix of Hampshire,United Kingdom), probed with 1:10 dilution of purified anti-MIWIantibody, and detected by anti-rabbit IgG antibodies conjugated withalkaline phosphatase or horseradish peroxidase. Immunohistochemicalstaining was performed on testis cryosections following the protocol ofAnderson et al (1999). Anti-MIWI antibodies, anti-TSX antibody(Cunningham et al., 1998), and anti-laminin antibody (Sigma Co. of St.Louis, Mo.) were used at 1:1, 1:1000, and 1:30 dilutions, respectively.The rat monoclonal antibodies BC7 and TRA54 (gifts from Dr. Y.Nishimune) were used at a dilution of 1:500, and monoclonal antibody EE2(also a gift from Dr. Y. Nishimune) was used at a dilution of 1:200. Allsecondary antibodies were purchased from Jackson ImmunoResearchLaboratory (West Grove, Pa.) and used at 1:100 dilution. DAPI (1 μg/ml)was used to label nuclei in some samples.

[0358] Construction of miwi^(null) and miwi^(Δc/Δc) mice. To generatethe miwi^(null) allele, the replacement targeting vector KO-4A wasconstructed in the pPNTIoxPneo vector (Shalaby et al., 1995) using a 2.0kb ApaI fragment and a 7.0 kb KpnI-NotI fragment for the 5′ and 3′flanking homologous regions, respectively (FIG. 11A). 25 μg ofNotI-linearized KO-4A DNA was used for electroporation of 1×10⁷ R1embryonic stem cells. 142 clones which survived the double selectionswere screened by PCR using primers 5′-ATGGGGTCTTTTCTTGCTCA-3′ (SEQ IDNO:14), and 5′-TGCCCATTAACATCACCATC-3′ (SEQ ID NO:15). The positiveclones were verified by Southern analysis using 3′ and 5′ externalprobes (FIG. 11B). Three recombinant embryonic stem cell clones (A9, E4,and G10) were injected into host blastocysts following standard,protocols (Hogan et al., 1994). One injected clone, A9, showed germlinetransmission of the mutant allele. The male chimera was crossed withC57BL/6J (JAX). Genomic DNA from the agouti F1 pups was analyzed by PCRfor the presence of the mutant allele using the following 3 primers(FIG. 11C): Primer a: 5′-TGATTTGGGGACTTATTTTAGAGC-3′ (SEQ ID NO:16),primer b: 5′-ACTTACCTTGTGACTTGGATGTG-3′ (SEQ ID NO:17), and primer c:5′-TTGAAAAGCATTGMCACCATAAG-3′ (SEQ ID NO:18). Homozygous mutant micewere obtained by crossing heterozygous males to either heterozygousfemales or homozygous mutant females. A miwi^(null) mouse colony ismaintained in a mixed genetic background of 129 and C57BL/6.

[0359] To generate the miwi^(ΔC) allele, the replacement targetingvector KO-5B was also constructed in the pPNTloxPneo vector (Shalaby etal., 1995) using a 1.9 kb XbaI-SalI fragment and a 7.0 kb KpnI-NotIfragment for the 5′ and 3′ flanking homologous regions, respectively(FIG. 12A). 25 μg of NotI-linearized KO-5B DNA was used forelectroporation of 1×10⁷ R1 embryonic stem cells. 130 clones thatsurvived the double selections were screened by PCR using primers5′-GATAACCACCGCCCTGCCTTTCAC-3′ (SEQ ID NO:19) and5′-TGCCCATTAACATCACCATC-3′ (SEQ ID NO:20). The positive clones wereverified by Southern analysis using a 3.6 kb Sac I genomic fragmentspanning exons 9-14 as the 5′ external probe and the same 3′ externalprobe as for the KO-4A construct (FIG. 12B). Three recombinant embryonicstem cell clones (A1, C10, and D2) were injected into host blastocystsfollowing standard protocols (Hogan et al., 1994). Two injected clones,C10 and D2, showed germline transmission of the mutant allele. The malechimera was crossed with C57BL/6J (JAX). Genomic DNA from the agouti F1pups was analyzed by PCR for the presence of the mutant allele using thesame method as for KO-4A. Homozygous mutant mice were obtained bycrossing heterozygous males to either heterozygous females or homozygousmutant females. A miwi^(ΔC) mouse colony is maintained in a mixed 129and C57BL/6 background as for miwi^(null).

[0360] Histology and cell proliferation/apoptosis assays. Male mice wereinjected intraperitoneally with 50 μg/g BrdU (bromodeoxyuridine) (RocheMolecular Biochemicals of Basel, Switzerland) 8 hours before beingsacrificed (6 hours for 2 week- and 4 week-old animals). Testes weredissected, weighed, fixed in either Bouin (Sigma of St. Louis, Mo.) or4% paraformaldehyde overnight at 4° C., and cut into 8 μm cryo- orparaffin sections. Hematoxylin/eosin staining and periodicacid-Shiff/hematoxylin staining were performed for histological analysisof paraffin sections. RNA in situ hybridization and immunohistochemicalstaining were generally performed on cryosections, and alternatively onparaffin sections. Incorporated BrdU signal was detected using a 1:3.5dilution of mouse anti-BrdU mAb (Becton, Dickinson, and Company ofFranklin Cakes, N.J.) and a FITC-conjugated goat anti-mouse secondaryantibody. Apoptosis was analyzed by TUNEL labeling (TdT-mediateddUTP-digoxigenin end-labeling) using the APOTAG® kit (Intergen ofPurchase, New York).

Results

[0361] Molecular cloning and mapping of miwi. To investigate thefunction of piwi family genes in mammalian development, a 4.06 kb miwicDNA containing a 191 bp 5′ untranslated region (UTR) was isolated. AcDNA containing 2.59 kb open reading frame (ORF), and a 1.17 kb 3′ UTRfollowed by a poly(A) tail was also isolated. The ORF is preceded bymultiple in-frame stop codons, indicating its completeness. Tocharacterize the miwi gene structure, about 40 kb of genomic DNA fromthe miwi locus was isolated. The miwi cDNA corresponds to a 19.2 kbgenomic region, encoded by 22 exons, with the ORF starting in the thirdexon and ending in the 22nd exon (FIG. 5). The previously defined PIWIbox is encoded by exons 20 and 21 (Cox et al., 1998). A representativemiwi cDNA is set forth as SEQ ID NO:3, and a representative MIWI proteinis set forth as SEQ ID NO:4.

[0362] To locate the miwi locus in the mouse genome, single strandpolymorphic mapping was performed against the European CollaborativeInterspecific Backcross panels. The mapping data indicated that miwi islocated on chromosome 5 near the D5nds6 marker with a lod score of 9.8.

[0363] The miwi ORF predicts that the MIWI protein contains 862 aminoacid residues (FIG. 6), with a relative molecular mass (M_(r)) of 98,600and an isoelectric point of 9.46. Except for a 100-200 amino acidstretch at the N-terminus, MIWI shares significant homology over itsentire length with other PIWI family proteins, such as MILI from mice(GenBank GI No.7416113; 42% identity), HIWI (Cox et al., 1998; 94%identity) and HILI (GenBank GI No. 8922370; 46% identity) from humans,PIWI (Cox et al., 1998; 37% identity) and AUBERGINE (Wilson et al.,1996; Schmidt et al., 1999; 38% identity) from Drosophila, PRG-1 andPRG-2 from C. elegans (Cox et al., 1998; 34% and 33% identity), and PAPfrom Paramecium (GenBank GI No. 6630673; 28% identity, respectively).Interestingly, all these proteins are either involved in the developmentof the germline or its equivalent. The homology at the C-terminal PIWIBox region is particularly high (FIG. 6), suggesting the potentialimportance of this region for the MIWI protein function.

[0364] miwi is specifically expressed in the germline duringspermatogenesis.

[0365] To explore the role of miwi in development, miwi expression wasassessed in major adult organs and in embryos of various stages byNorthern blot analysis. A Northern blot was prepared using adult mousetissues, including heart, brain, spleen, lung, liver, muscle, kidney,and testis, and hybridized using a miwi probe. miwi encodes a 4.7 kbabundant transcript and a 6.5 kb present at low levels, both of whichare specifically expressed in the adult testis, but are not detectablein other adult organs. Hybridization with a β-actin probe was performedas a loading control. A Northern blot was also prepared using stagedembryos at 7, 11, 15, and 17 days post coitum (dpc) and hybridized usinga miwi probe. miwi transcript was not detected in embryos at any of thestages examined. The adult- and testis-specific expression of miwi wasfurther confirmed by in situ hybridization, by examining the expressionof the miwi-GFP knock-in chimeric gene, and by Western blot analysis(described herein below).

[0366] A Northern blot was also prepared using staged embryos at 7, 11,15, and 17 days post coitum (dpc) and hybridized using a miwi probe.miwi transcript was not detected in embryos at any of the stagesexamined.

[0367] miwi RNA expression was further examined in testis using tissuesections multiply labeled to detect miwi RNA and the laminin antigenfound on seminiferous tubules. miwi RNA and laminin were distinguishedby use of alternate fluorescent labels. In the testis, miwi RNA is notdetectable at 1, 3, 6, and 9 days post partum (dpp), but is firstdetected in cells in the center of the seminiferous tubules at 12 dpp, astage when zygotene spermatocytes first appear. When the tubule lumenbecomes conspicuous at 14 dpp, miwi RNA is restricted to the lumenalside of the seminiferous epithelium where developing spermatocytesreside. In fully developed adult testes, miwi RNA is detected in a smallnumber of germ cells at the basal layer of the tubule, and is abundantlypresent in primary spermatocytes that are one-cell layer away from thebasement membrane.

[0368] The germline-specific expression of miwi was confirmed bycounter-staining testis sections with the anti-TSX (testis-specificX-linked gene product) antibody, a Sertoli cell marker (Cunningham etal., 1998). The miwi-expressing cells do not express TSX, suggestingthat they are not Sertoli cells. At all postnatal stages, miwi RNAexpression is only detectable in a subset of seminiferous tubules,indicating that miwi expression is dependent on seminiferous tubulecycle.

[0369] MIWI is a cytoplasmic protein. To further characterize miwifunction, anti-MIWI antibodies were generated that recognize an about100 kD antigen of expected MIWI protein size. For detection of MIWIprotein in adult tissues, a Western blot was prepared using adultdissected mouse tissues, including bone marrow, brain, heart, kidney,liver, lung, ovary, spleen, testis, and thymus. 50 μg of protein wereloaded in each lane, and the blot was probed using an anti-MIWIantibody. The MIWI antigen is detectable only in the testis, where it isexpressed specifically in the germline in a pattern similar to the miwiRNA expression pattern. Moreover, this antigen is eliminated inmiwi^(ull) testes, as indicated by Western blotting and byimmunofluorescence microscopy analyses (as described herein below).These data indicate that the antigen detected by the anti-MIWI antibodyis the MIWI protein.

[0370] To further evaluate the distribution of MIWI protein in testis,immunostaining experiments were performed to simultaneously detect MIWIprotein and laminin, and further stained using DAPI. The MIWI andlaminin labels were detected using different fluorophores such thatMIWI, laminin, and DAPI were each distinguishable when viewed using afluorescence microscope.

[0371] MIWI is a cytoplasmic protein that is first detectable at 0.14dpp, soon after the first appearance of zygotene stage spermatocytes. Inthe adult testis, MIWI is detected in primary spermatocytes 1-2 celllayers away from the basal lamina. The level of MIWI protein is sharplyreduced in secondary spermatocytes and round spermatids, and is notdetectable in elongating spermatids or mature sperm. In addition, MIWIis not detectable in somatic cells such as Sertoli cells or interstitialcells outside the tubules. This expression pattern suggests that MIWIcan play a cell autonomous role in spermatocyte development and/orspermatid differentiation.

[0372] Spermatogenesis in miwi^(null) mice is arrested at the onset ofspermiogenesis. To investigate the function of MIWI in vivo,miwi-deficient mice were generated by homologous recombination thatreplaced the genomic region corresponding to almost the entire MIWI ORFwith a MmGFP ORF (Zernicka-Goetz et al., 1997) (FIG. 11A). The resultingrecombinant allele contains a fusion ORF that encodes the first nineamino acid residues of MIWI, followed by a PPRQ linker (SEQ ID NO:21)and the MmGFP peptide. This genotype was confirmed by genomic Southernanalysis (FIG. 11B) and PCR genotyping analysis (FIG. 11C). In thesehomozygous miwi-deficient mice, no native MIWI protein was detected inthe testis by Western blot analysis (FIG. 11D) or by immunofluorescencemicroscopy. Thus, this allele was designated miwi^(null).

[0373] The miwi^(null) mice, also referred to as miwi^(−/−) mice,develop to apparently normal adults. Interbreeding of heterozygotesmiwi^(−/−) adults yielded the Mendelian ratio (71: 139: 63) of wild typemiwi^(+/−, heterozygous miwi) ^(+/−), and homozygous miwi^(−/−)offsprings. In addition, crossing heterozygous miwi^(+/−) males tohomozygous miwi females also yielded the predicted 1:1 ratio ofheterozygous miwi to homozygous miwi^(−/−) offspring (83:75). Theseresults indicate that there is no lethality caused by the miwi^(null)mutation. Furthermore, miwi^(null) mice are female fertile butcompletely male sterile. Testes from adult miwi^(−/−) males (8-24 weeksold, n=28) are on average 29% smaller by weight than wild type ormiwi^(−/−) testes (n=28). Moreover, no sperm is found in the epidydimusof miwi^(−/−) mice.

[0374] To evaluate spermatogenesis in miwi^(−/−) mice, testis sectionswere procured from miwi^(−/−) and miwi^(−/−) mice, and stained withhematoxylin/eosin to reveal cellular morphology and enable theidentification of cell types including spermatogonia, spermatocytes,round spermatids, and elongated spermatids. Histological examination ofmiwi^(−/−) testes revealed that spermatogenesis is arrested at the roundspermatid stage. Spermatids fail to initiate cellular elongation ornuclear condensation (step 8 of spermiogenesis). In some tubules, evenround spermatids are absent.

[0375] Testis sections were also immunostained to detect cyclin D3, anantigen strongly expressed in elongated spermatids (Zhang et al., 1999).In contrast to abundant staining in miwi^(+/−) testes, cyclin D3 was notdetected in miwi testes.

[0376] To further characterize the miwi phenotype, testis sections werelabeled with an antibody that specifically binds the TRA54 antigen,which labels a round organelle in pachytene spermatocytes and theacrosome in spermatids before step 12 of spermiogenesis (Pereira et al.,1998). In testes derived from miwi^(−/−) mice, TRA54 staining wasdelocalized or absent, indicating abnormal or absent acrosomes. Periodicacid-Schiffs staining of testis sections Was also performed, and did notdetect any acrosomes in miwi^(−/−) round spermatids.

[0377] Collectively, the histological and immunostaining observations oftestis development in miwi^(−/−) mice indicate that miwi is requiredduring the initial stages of spermiogenesis.

[0378] miwi^(null) mice show early spermatogenic defects. Prior toarrest at the round spermatid stage, miwi^(−/−) mice show earlierspermatogenetic defects. Degenerating germ cells with pycnotic nucleiwere frequently observed in regions corresponding to spermatocytes andspermatids. TUNEL labeling (TdT-mediated dUTP-digoxigenin nickend-labeling) revealed that miwi^(−/−) testes contain a drasticallyincreased number of apoptotic cells from the basal layer to the lumenallayer of the seminiferous epithelium, when compared to the number ofapoptotic cells in a same testes region derived from a miwi^(+/−) mouse.TUNEL labeling was observed in testis sections also labeled to detectlaminin to outline the seminiferous tubules and with DAPI, to facilitatedetermination of the position and identity of dying cells. The resultssuggest that miwi is required for the survival and development ofspermatogonia, spermatocytes, and round spermatids.

[0379] A related observation is that antibody EE2, which recognizes thespermatogonia and early spermatocytes at the basal layer (Koshimizu etal. 1995; up to zygotene stage), frequently marks two layers of germcells in miwi^(−/−) testes. Testis sections were viewed when multiplylabeled to detect the EE2 antigen, laminin, and nuclei. The EE2 andlaminin staining protocols employed different fluorophores such thateach label was distinguishable from the other, and furtherdistinguishable from the nuclear dye DAPI.

[0380] To distinguish whether expansion of the germ cell layer was dueto overproliferation or delayed differentiation of spermatogonia and/orearly spermatocytes, the proliferation of germ cells was examined usinga BrdU (bromodeoxyurdine) incorporation assay. miwi^(+/−) and miwi^(−/−)testes were observed to incorporate BrdU to a similar extent, indicatinga similar frequency of cell division. Hence, the miwi deficiency appearsto cause delayed differentiation rather than overproliferation.

[0381] To further distinguish whether a differentiation delay occurs inspermatogonia or in spermatocytes, anti-BC7 was used to labelspermatocytes from leptotene to early pachytene stages (Koshimizu etal., 1993). Testis sections were viewed when multiply labeled to detectthe BC7 antigen, laminin, and nuclei. The BC7 and laminin stainingprotocols employed different fluorophores such that each label wasdistinguishable from the other, and further distinguishable from thenuclear dye, DAPI miwi^(+/−) and miwi^(−/−) testes showed a similarpattern of BC7 staining, suggesting that the delay occurs at thespermatogonial stage. Thus, miwi deficiency appears to cause a delay inspermatogonial differentiation.

[0382] To examine when these defects first occur during postnataldevelopment in miwi^(−/−) animals, testis histology was examined atvarious postnatal stages.

[0383] Histological review was facilitated by staining of testissections with haemotoxylin/eosin. Although there was no detectabledifference between miwi^(+/−) and miwi^(−/−) testes from 2-week oldmice, the spermatogenic defects described above were evident in 4-weekold miwi^(−/−) testes. This time point coincides with the firstappearance of elongated spermatids in wild type and heterozygous testes.These observations suggest that in miwi^(−/−) testes spermatid arrestoccurs during the first wave of spermatogenesis while defects inspermatogonia and spermatocytes are manifested in a more gradualfashion.

[0384] Deleting the C-terminal PIWI box region of MIWI causesmiwi^(null)-like defects. The piwi family genes are highly conserved inthe C-terminal PIWI box-coding region (Cox et al., 1998) (FIG. 6). Theessential role of miwi in spermatogenesis provides an opportunity totest whether the PIWI box-coding region is important for MIWI function.miwi-deficient mice were generated in which the C-terminal 245 aminoacid residues of MIWI are replaced by a 239 amino acid residueMmGFP-coding sequence (FIG. 12A). The resulting recombinant miwi^(ΔC)callele contains a fusion ORF that encodes the first 617 amino acidresidues of MIWI fused to the 239 residue MmGFP peptide, generating aM_(r) 97,600 hybrid protein that is essentially the same size as theMIWI protein (M_(r) 98,600). This genotype was confirmed by genomicSouthern and PCR genotyping analyses. Indeed, the hybrid protein ispresent in homozygous miwi^(ΔC) mice, suggesting that substituting theC-terminal 245 residues of MIWI with a MmGFP peptide of similar lengthdoes not significantly affect the stability of the protein (FIG. 12B).

[0385] Homozygous miwi^(ΔC) mice show phenotype that areindistinguishable from that of homozygous miwi^(null) mice. First, themiwi^(ΔC) mice develop normally to adulthood at expected Mendelianratios. Intercrosses between miwi^(ΔC/+) mice produced the expectedMendelian ratios of progeny genotypes, i.e, 17 miwi^(+/+), 46miwi^(ΔC/+), and 23 miwi^(ΔC/ΔC) mice. Second, miwi^(ΔC/ΔC) mice werefemale fertile but completely male sterile. Third, histologicalexamination of testes derived from miwi^(ΔC/ΔC) revealed thatspermatogenesis is also arrested at the round spermatid stage, beforestep 8 of spermiogenesis. No spermatid was ever detected to undergocellular elongation or nuclear condensation. In some tubules, even roundspermatids are absent. Fourth, prior to arrest at the round spermatidstage, miwi^(ΔC/ΔC) mice showed earlier spermatogenic defectsindistinguishable from those observed in miwi^(null) animals, asassessed by histological analysis at three weeks. Degenerating germcells with pycnotic nuclei were frequently observed in regionscorresponding to spermatocytes and spermatids. Lastly, the earlyspermatogenic defects were first detectable in miwi^(ΔC/ΔC) at 21 dpp.Similarly, miwi^(null) mice show spermatogenic defects by 28 dpp, butare not observed at 14 dpp. The apparently identical phenotypes ofmiwi^(null) and miwi^(ΔC/ΔC) mice suggest that the C-terminal PIWIbox-containing region is essential for MIWI function.

Discussion

[0386] Although a number of genes are known to be involved inspermatogenesis, only a few such genes are known to have testis-specificexpression or function. As disclosed herein, miwi is essential forspermatogenesis, and miwi is expressed specifically in the testiculargermline. The phenotype of miwi null mutant mice reveals the involvementof miwi in both early and late stages of spermatogenesis, while thegermline-specific expression of miwi further suggests that MIWIfunctions cell-autonomously during mammalian spermatogenesis. Since miwiis a member of the piwi gene family, the disclosure herein reveals, forthe first time, the novel function of this gene family in mammaliansystems.

[0387] The role of MIWI as a cell-autonomous molecule essential forspermiogenesis. Mammalian spermatogenesis is controlled by both inter-and intracellular mechanisms. The intercellular mechanism includes BMP8aand b (bone morphogenetic proteins 8a and 8b) signaling, which isrequired for the initiation and maintenance of spermatogenesis, withtheir downstream factor MADR1 (MAD-related protein 1) expressed in thegermline (Zhao et al., 1996; Zhao and Hogan, 1997; Zhao et al.; 1998).In addition, DHH (desert hedge hog) signal from Sertoli cells isrequired for various stages of spermatogenesis apparently via regulatingthe formation of adult-type Leydig cells and the normal development ofperitubular cells and seminiferous tubules (Bitgood et al., 1996; Clarket al., 2000). Moreover, STEEL (stem cell factor) signaling from Sertolito the c-KIT receptor (receptor tyrosine kinase) on the surface of germcells is required for spermatogonial differentiation (Yoshinaga et al.,1991; Ohta et al., 2000).

[0388] By contrast, germline-specific expression of miwi suggests thatMIWI acts cell-autonomously in spermatogenesis. This mode of action isshared by cell cycle regulators such as cyclin A1 and HSP-70.2 (heatshock protein of 70.2 kD) (Dix et al., 1996; Zhu et al., 1997; Liu etal., 1998), DNA repair and recombination genes such as MLH1 (MutLhomolog 1), MSH5 (muscle segment homeobox 5), ATM (ataxia telangiectasiamutated), SCP3 (synaptonemal complex protein. 3) (Baker et al., 1996;Barlow et al., 1996; Edelmann et al., 1996; Elsbn et al., 1996; Xu etal., 1996; de Vries et al., 1999; Edelmann et al., 1999; Yuan et al.,2000), transcription factors such as A-MYB (MYB-related protein A) andEGR4 (early growth response protein 4) (Toscani et al., 1997;Tourtellotte et al., 2000), and RNA binding proteins such as DAZLA(deleted in azoospermia-like) and VASA (Ruggiu et al., 1997; Tanaka etal., 2000). However, targeted disruptions of these additional genesblock spermatogenesis at the spermatocyte stage rather than the roundspermatid stage, as observed in miwi^(−/−).

[0389] The miwi phenotype is similar to the effect of prematuretranslation of protamine-1 in round spermatids (Lee et al., 1995).However, no protamine is detected in miwi^(null) mice, indicating thatthe premature accumulation of protamines is not the cause of thespermatogenic arrest in these mice.

[0390] A molecule whose deficiency causes miwi^(null)-like arrest at theround spermatid stage is CREM (cAMP responsive element modulator), atranscription factor proposed to be a master switch for spermiogenesis(Blendy et al., 1996; Nantel et al., 1996; Zhou et al., 1996;Sassone-Corsi 2000). CREM is essential for the expression ofspermiogenic factors such as protamines, transition proteins (TNPs),calspermine, mitochondrial capsule selenoprotein andangiotensin-converting enzyme. The phenotypic similarity between miwiand CREM mutants suggests that miwi could be involved in initiatingspermiogenesis as well. This “master switch” phenotype differs from thatof other spermiogenic factors, such as TNP1 (transition protein 1)involved in histone displacement and chromatin condensation (Yu et al.,2000), the RNA binding protein TARBP2 (TAR (HIV) binding protein 2) thatregulates protamine translation (Zhong et al., 1999), HR6Bubiquitin-congugating enzyme involved in DNA repair (Roset et al.,1996), or casein kinase required for nuclear condensation and acrosomedevelopment (Xu et al., 1999). Loss of TNP1, TARBP2, HR6B, or caseinkinase function causes limited abnormalities at the elongated spermatidstage, but spermiogenesis is nevertheless completed at observablefrequencies. The definitive arrest of spermatogenesis at the onset ofspermiogenesis in miwi mutants suggests that MIWI is essential for theinitiation of the entire spermiogenic program.

[0391] The role of MIWI in early spermatogenesis. In addition to itsessential role in spermiogenesis, miwi appears to be involved in earlierstages of spermatogenesis, even as early as the germline stem cellstage. An earlier role is suggested by the delay of spermatogonialdifferentiation and elevated apoptosis in spermatogenic cells in miwimutant testes, as well as by abundant miwi expression in spermatocytes.

[0392] Molecular activity of MIWI. All PIWI family proteins share ahighly conserved C-terminal region that is enriched with highly basicand highly charged residues (Cox et al., 1998). As disclosed herein,replacement of the C-terminal region by an unrelated peptide sequence ofsimilar size (i.e. the GFP sequence) appears to completely abolish theMIWI activity in various stages of spermatogenesis, resulting in amiwi^(null) like phenotype. These results provide the first evidencethat this region is crucial for the function of a PIWI family protein.

[0393] In summary, miwi is essential for germline stem cell division andother stem cell-related processes. Thus, miwi and other mammalian piwifamily genes are useful in the therapeutic methods, screening methods,and diagnostic and prognostic methods of the present invention set forthherein.

Example 4 PIWI Family Proteins are Nuclear Proteins Whose LevelDetermines the Number of Stem Cells in the Germline

[0394] Two immunofluorescence micrographs were prepared and showedgermline cells (marked in green) in the Drosophila germarium in thebackground of normal and elevated level of PIWI. The ectopicallyelevated expression of piwi causes an increase in the number of stemcells and/or their immediate daughter cells (cystoblasts) from normally2-3 to approximately 15, with 7 of which were shown in the focal plan ofmicrograph of elevated PIWI levels. Stem cells were marked by thespectrosome (Sp), an organelle specific to these cells. Thedifferentiating germline cyst contains fusome (F), an organelle specificto mitotic stage of cyst development. Thus, over-expression of piwiincreases the number of germline stem cells/cystoblasts.

[0395] Immunofluorescence micrographs of detection of a myc-tagged PIWIprotein (with and without myc as a control) in Drosophila ovariole,Drosophila terminal filament, basal cells and GSC in Drosophilagermarium, apical polar cells (APC) in Drosophila egg chambers, and hubcells in Drosophila testis were also prepared. These micrographs showedthat PIWI is a nuclear protein present in both the germline and insomatic cells at different stages of oogenesis and spermatogenesis.myc-PIWI was shown to completely rescue the defects caused by loss ofpiwi function, thus demonstrating a therapeutic method of the presentinvention. PIWI is demonstrated to be a nuclear protein at certaindevelopment stages. MIWI and HIWI proteins are thus also contemplated tobe nuclear proteins at certain development stages.

[0396] Additionally, as disclosed below, the PIWI protein can localizeeither in the nucleoplasm or cytoplasm, the dose of the PIWI proteindetermines both the number and the mitotic rate of germline stem cells,and the PIWI protein is localized in the germplasm (the cytoplasm offuture germ cells) and plays a key role in determining the initialnumber of germ cells in during embryogenesis. The mouse PIWI protein(i.e., MIWI protein) has also been observed in the cytoplasm viaantibody screening and is thus also envisioned to be a cytoplasmicfactor with very similar functions in germ cell and stem celldetermination. Thus, the HIWI protein is thus also envisioned to be acytoplasmic protein at certain development stages.

Example 5 piwi Encodes a Nucleoplasmic Factor Whose Activity Modulatesthe Number and Division Rate of Germline Stem Cells

[0397] As disclosed above, piwi represents the first class of genesknown to be required for stem cell self-renewal in diverse organisms. Inthe Drosophila ovary, piwi is required in somatic signaling cells tomaintain germline stem cells. In this Example it is shown that that piwiencodes a novel nucleoplasmic protein present in both somatic andgermline cells, with the highly conserved C-terminal region essentialfor its function. Removing PIWI protein from single germline stem cellssignificantly decreases the rate of their division. This is indicativeof a second role for PIWI as a cell-autonomous promoter of germline stemcell division. Consistent with its dual function, over-expression ofpiwi in somatic cells causes an increase both in the number of germlinestem cells and the rate of their division. Thus, PIWI is a key regulatorof stem cell division—its somatic expression modulates the number ofgermline stem cells and the rate of their division while its germlineexpression also contributes to promote stem cell division in acell-autonomous manner.

Materials and Methods

[0398]Drosophila strains and culture. All Drosophila strains were grownat 25° C. on yeast-containing molasses/agar medium. The following flystrains were used in this study: piwi¹ and piwi² mutant chromosomes,generated in different genetic screens (Lin and Spradling, 1997), weredominantly marked with irregular facets (If (Lindsley and Zimm, 1992);ep(2)1024 is an EP element insertion (Rorth, 1996; Rorth et al., 1998)in the piwi 5′ UTR; piwi¹ FRT40A/CyO and piwi²FRT40A/CyO; hsFIp;+FRT40A/CyO (Xu and Rubin, 1993); hsFIp; armadillo-lacZ FRT40A (armlacZFRT40A, Lecuit and Cohen, 1997); hsGal4 (Brand and Perrimon, 1993) areas described herein above.

[0399] Generation of mutant germline stem cell clones. piwi mutant GSCclones were generated by Flp-mediated recombination as described by Xuand Rubin (1993). To generate wild-type and mutant GSC clones foranalyses, piwi⁺FRT40A/CYO, piwi¹FRT40A/CyO and piwi²FRT40A/CyO maleswere mated to w hsFlp1; armlacZ FRT40A virgin females, respectively, toproduce w hsFlp1/+; piwi* FRT40A/armlacz FRT40A progeny, where * denotesthe wildtype or mutant alleles. The parental adults were transferred tofresh vials after 2 days. Larvae from the original vials wereheat-shocked once daily for 1 hour in a 37° C. water bath on days 3 and4 to induce mitotic recombination immediately prior to the onset ofoogenesis at the late third instar stage as described herein above.After eclosion, adult w hsFlp1/+; piwi* FRT40A/armlacZ FRT40A femaleswere transferred to fresh food at room temperature and ovaries wereremoved 1 week, 2 weeks, and 3 weeks after the last heat shock treatmentand processed for anti-lacZ antibody staining to look for lacZ-negativeclones.

[0400] Heat-shock induced PIWI overexpression. To construct apiwi-overexpressing stock, ep(2)1024/CyO virgin females were crossed tomales homozygous for a hsGal4 transgene on chromosome 3. Femalescarrying both the ep(2)1024 chromosome and the hsGal4 transgene wereheat shocked at 37° C. for 1 hour each time with an interval of 12 hoursfor 3-4 days. At the end of the heat shock regime, ovaries weredissected and processed for antibody staining.

[0401] To analyze the functionality of the PIWI protein produced byhsGal4-induced overexpression of ep(2)1024, ep(2)1024/CyO; hsGal4/hsGal4virgin females were crossed to piwi¹If/CyO; +/+ males to produce theep(2)1024/piwi¹lf; hsGal4/+ transheterozygous progeny. After 2 days,parental adults were transferred to fresh vials. Larvae were heatshocked daily for 1 hour at 37° C. starting at day 3 and continued to 3days after eclosion. The transheterozygous progeny were subsequentlydissected and processed for antibody staining. The transheterozygousprogeny which were not subjected to the heat shock regime served ascontrols.

[0402] Immunofluorescence microscopy and BrdU labeling, Ovaries andtestes were dissected, fixed and stained as described by Lin et al.(1994). For immunofluorescence staining the following antisera wereused: polyclonal anti-Vasa antibody (1:2000; Hay et al., (1990)),monoclonal anti-Hts antibody 1 B1 (1:1; Zaccai and Lipshitz, (1996)),polyclonal anti-α-spectrin antibody (1:200; Byer et al., (1987)),monoclonal anti-myc epitope antibody 1-9E10.2 (1:50; Evan et al., 1985),monoclonal anti-BamC antibody (1:1000; McKearin and Ohlstein, 1995),polyclonal anti-β-galactosidase antibody (1:600; Cappel, Costa Mesa,Calif.), monoclonal anti-BrdU antibody (1:50; Becton-Dickinson,Franklin. Lakes, N.J.). All the fluorescence-conjugated secondaryantibodies were from Jackson Immunoresearch Laboratory (Westgrove, Pa.)and were used at 1:200 dilution.

[0403] Immuno-fluorescently labeled samples were also counter-stainedwith DAPI as described previously (Lin and Spradling, 1993). Micrographswere taken using either a Zeiss AXIOPLAN® microscope or a Zeiss LSM410™confocal microscope (Zeiss, Oberkochen, Germany) as described hereinabove. BrdU labeling was performed essentially as described in Gonczyand DiNardo (1996). Briefly, on day 3 of the heat shock regime,ep(2)1024; hsGAL4 females were transferred to an Eppendorf tube that washeld horizontally and contained 100 μl of 100 mM BrdU in grape juice.The flies were fed (pulse) with BrdU for 1 hour at room temperature.After the pulse, flies were returned to fresh vials without BrdU for 12hours at room temperature (chase). These females were subsequentlydissected and fixed for immunofluorescence labeling with anti-BrdU andanti-α-spectrin antibodies.

[0404] Construction of transgenes encoding N- and C-terminal-tagged PIWIproteins. Synthetic oligonucleotides (Gibco BRL) encoding a myc-epitopewere cloned into unique sites at the N- and C-terminus of the piwi ORFin a 6.8 kb piwi genomic construct (pRc12) that fully rescues the piwiactivity (Examples 1-4 above and Cox et al., 1998) as follows: For theN-terminal myc insertion, the following primers were used: Bcl/myc:5′-GAT CAT ATG GAG CAA AAG CTT ATT AGC GAG GM GAT CTG MT-3′ (SEQ IDNO:7) and Bcl/antisense myc: 5′-GAT CAT TCA GAT CTT CCT CGC TAA TAA GCTTTT GCT CCA TAT-3′ (SEQ ID NO:8). The primers were annealed according tothe manufacturer's recommendations and cloned into a unique BcIlrecognition site in a Bluescript clone containing piwi genomic DNA.

[0405] The resulting recombinant myc-piwi gene was cloned into pCasper 4(Pirrotta, 1988) to produce the plasmid pPMB1-6 in which the mycsequence is inserted between PIWI amino acid residues 3 and 4. For theC-terminal myc insertion, the following primers were used: Nhelmyc:5′-CTA GCA TAT GAG CAA AAG CTT ATT AGC GAG GAA GAT CTG AAT AAG-3′(SEQ.ID NO:9) and Nhe/antisense myc: 5′-CTA GCT TAT TCA GAT CTT CCT CGCTAA TAA GCT TTT GCT. CAT ATG-3′ (SEQ ID NO:10). Primers were annealed asdescribed above and cloned into a unique NheI recognition site in aBluescript clone containing part of piwi genomic DNA. The recombinantinsert was cloned into pCasper 4 (Pirrotta, 1988) to produce the plasmidpPMN in which myc is inserted between PIWI amino acid residues 795 and796. The insertions were confirmed by DNA sequencing.

[0406] P-element mediated germline transformation. Transgenic flies wereproduced according to Spradling and Rubin, 1982, using w; 2-3 Sb e/TM6 e(Robertson et al., 1988) embryos as recipients. Eight independentpPMB1-6 lines and twelve independent pPMN lines were recovered. Themyc-tagged transgenes were then separated from the 2-3 transposase andintroduced into the homozygous piwi¹ background for fertility tests bygenetic crosses. Rescue crosses were carried out at 25° C. Eachtransgene was further tested for Myc expression by whole-mountimmunofluorescence.

[0407] Immunoblotting analysis and in vitro translation. To preparetotal protein extracts from pPMB1-6 transgenic flies, ovaries weredissected in 1×EBR (1.30 mM NaCl; 5 mM KCl; 2 mM CaCl₂; 10 mM Hepes pH6.9) and homogenized in 1× sample buffer (5% 2-mercaptoethanol, 3% SDS,10% glycerol, 62.5 mM TrisCI pH 6.8, 0.1% bromophenol blue). Proteinsamples were boiled 5 minutes at 95° C. and then loaded on 10% SDS-PAGEgels. After electrophoresis, gels were electro-transferred to Genescreen(NEN Research Products, DuPont, Wilmington, Del.) in a TRANS-BLO®apparatus (Bio-Rad Laboratories, Inc., Hercules, Calif.). Blots wereblocked for 1 hour in Blotto (5% non-fat dry milk in 1×PBS) and primaryantibodies were subsequently diluted in Blotto followed by an incubationovernight at 4° C. The monoclonal 9E10 anti-myc antibody (Evan et al.,(1985)) was used at a dilution of 1:50. HRP-conjugated anti-mouse IgGsecondary antibody (Jackson Immunoresearch Laboratory, Westgrove, Pa.)was used at 1:4000. Blots were washed and processed for detection usingenhanced chemiluminescent. (ECL) reagents as recommended by themanufacturer (Amersham, Buckinghamshire, England). In vitro translationanalysis was performed on the plasmid pDC2 to produce ³⁵S-labeled PIWIprotein according to the manufacturer's recommendations (Promega,Madison, Wis.). The translation products were separated on a 10%SDS-PAGE gel and images were collected by exposing the gel to X-rayfilm.

[0408] Quantitation of germline stem cell division rate. Relativedivision rate of piwi⁻ GSCs was determined as follows: The number ofmarked piwi⁻ cysts in 30 germaria was counted to obtain the averagenumber of the piwi⁻ cysts per germarium. This number is then divided bythe average number of marked wild-type cysts per germarium as obtainedfrom counting 20 germaria. The relative division rate of GSCs in thepiwi-overexpressing germaria and that in the wild-type and un-inducedep(2)1024 control germaria was determined by counting the frequency oftelophase spectrosomes contacting the cap cells in the 2-3 GSCs in themost apical region of the germarium.

Results

[0409] PIWI is a nuclear protein present in both the soma and germlinein both sexes. Examples 14 above demonstrate that piwi encodes a 97.2kDa novel protein required for the self-renewing division of GSCs inboth males and females. To elucidate further the function of piwi inregulating GSC division, the expression and subcellular behavior of thePIWI protein was studied in vivo. The PIWI protein was tagged byinserting a sequence encoding a myc epitope (Evan et al., (1985)) intothe piwi gene, at the 5′ end of the piwi open reading frame (FIG. 2).The p[5′-myc-piwi] transgene (denoted as myc-piwi) was introduced intoDrosophila via P-element mediated germline transformation (Spradling andRubin, (1982)). myc-piwi fully restores the fertility and gametogenesisof piwi¹ mutant males and females (Table 4). Thus, the myc-PIWI proteinconfers wild-type PIWI function. In contrast, a p[piwi-3′-myc] transgene(denoted as piwi-myc) with the myc sequence inserted in the highlyconserved C-terminal region (FIG. 7) fails to rescue any piwi mutantphenotype (Table 4). The PIWI-myc protein was not detectable either byWestern blotting, analysis or by immunostaining of the transgenicovaries (Table 4), indicating that this region is essential at least forthe stability of the PIWI protein. TABLE 4 Rescue of the piwi¹ phenotypeand myc expression by myc-PIWI and PIWI-myc¹ rescue of piwi¹ fertility²# of independent Staining female male transgenic lines for myc piwi¹myc; piwi¹ piwi¹/CyO piwi¹ myc; piwi¹ piwi¹/CyO myc-PIWI  8 100% (8) 034.9 ± 6.8 30.9 ± 8.7 0 49.8 ± 6.7 49.8 ± 0.6 PIWI-myc 12  0% (7) 0 030.9 ± 8.7 0 0 49.8 ± 0.6 # piwi¹/piwi¹ transgenic lines. Homozygouspiwi¹ females not only show zero fertility as indicated, but also failto lay eggs. The small number of eggs produced by these females areretained in the ovary and eventually undergo atrophy.

[0410] The expression of myc-PIWI was confirmed by immunoblot analysisusing a monoclonal anti-myc antibody as a probe, which reveals a single97.5 kDa band in the ovarian extract of myc-piwi flies. This agrees wellwith both the 97.2 kDa predicted size of PIWI and the 97.1 kDa observedsize of PIWI when a full-length piwi cDNA was translated in vitro.Furthermore, eight independent myc-piwi transgenic lines share theidentical subcellular localization pattern throughout gametogenesis.Thus, the myc-PIWI protein reflects the function and distribution ofendogenous PIWI.

[0411] The expression and localization pattern of myc-PIWI duringgametogenesis was also examined by immunofluorescence microscopy. Wholemount ovaries and testes isolated from wild-type flies carrying amyc-piwi transgene were stained with the anti-myc antibody to visualizemyc-PIWI; this antibody did not detect any signal above the backgroundin ovaries and testes from siblings without myc-piwi. The samples werealso stained with anti-VASA antibody to highlight germ cells and DAPI tolabel nuclear DNA. In the third instar larval ovary, where GSCs begintheir asymmetric divisions, myc-PIWI was found in the nucleus of allgerm cells of the ovary. In addition, myc-PIWI was also present in thenuclei of the forming terminal filament cells. In adult ovaries,myc-PIWI is present in the nuclei of both the, somatic and germlinecells, as predicted by piwi RNA in situ analyses described in Examples1-4. Specifically, in the germarium, myc-PIWI is expressed in all thesomatic cells, including the main terminal filament cells, the cap cellsand the inner sheath cells, consistent with the genetic clonal analysespresented herein above which indicated that piwi is required in thesecells to maintain GSCs. PIWI is also expressed in somatic stem cells andthe follicle cells in the germarium.

[0412] In the germline, myc-PIWI shows a dynamic nuclear expressionpattern: it is present abundantly in GSCs, but is down-regulated-incystoblasts and developing cysts. In 16-cell cysts in regions 2 and. 3,the myc-PIWI regains its high level expression, and remains so in nursecells and oocytes in post-germarial egg chambers throughout oogenesis.This expression is consistent with the clonal and RNA in situ analysespresented herein above which indicate that PIWI is also loaded in eggsas a maternal component essential for embryogenesis. In allpost-germarial egg chambers, myc-PIWI is also specifically expressed inthe anterior polar follicle cells. The consistent nuclear localizationof myc-PIWI in various types of somatic and germline cells duringoogenesis indicates that PIWI is a nuclear protein.

[0413] Therefore, PIWI is a nuclear protein present in the soma andgermline of both sexes. In the third instar larval ovaries, PIWI ispresent in the nucleus of all germline cells (GSC) as well as in formingterminal filament cells (TFC) as revealed by myc-PIWI in red and byVASA, a germline-specific marker, in green. PIWI is expressed in adynamic pattern in the adult germarium (Ge), while being weaklyexpressed in main terminal filament cells and strongly expressed in capcells (CpC). In the soma, PIWI is strongly expressed in the cap cells,inner sheath cells (ISC), somatic stem cells (SSC), and follicle cells(FC). In the germline, PIWI is expressed in germline stem cells (GSC)and is downregulated in cystoblasts (Cb) and early mitotic cysts. PIWIregains its high level expression in differentiating 16-cell cysts (DC)in regions 2b and 3.

[0414] In post-germarial egg chambers, PIWI is expressed strongly in allnurse cell nuclei (NCN) as well as in the nucleus (germinal vesicle, GV)of the developing oocyte. In the somatic follicle cell layer around eachegg chamber, PIWI is expressed in anterior polar follicle cells (APC).The sharp down regulation of PIWI in cystoblast is also evident in thegermarium, in which the three PIWI-bright small nuclei at the germarialapex are cap cell nuclei. The two larger nuclei in the next layer areGSC nuclei. Other small PIWI-bright nuclei in region 1 are inner sheathnuclei. The PIWI-negative region starts with a cystoblast positioned inthe third layer.

[0415] In the third instar larval testis, PIWI, is present in the nucleiof both somatic and germline cells. In the soma, PIWI is expressed inthe nuclei of hub cells (Hub) apical to GSCs and in cyst progenitorcells (CPC). In the germline, PIWI is present in the nuclei of GSCs andtheir immediate daughters. The apical region of the adult testis showsthe same apical-distal organization as the third instar larval testis.PIWI expression pattern in the adult testis is identical to that in thethird instar larval testis. PIWI is expressed in hub cells, GSC, andCPC. Ovaries from sibling females which do not carry the myc-piwitransgene show no specific staining with the anti-Myc antibody.

[0416] PIWI is also expressed as a nuclear protein duringspermatogenesis. In the third instar larval testes which contain mostlypremeiotic germ cells, myc-PIWI is localized to the nuclei of apicalsomatic cells, including the hub cells, which are the testicularequivalent of terminal filament cells. myc-PIWI is also present insomatic stem cells and their progeny, the cyst progenitor. At the apicalgermline, PIWI is present in the nuclei of GSCs and their immediatedaughter cells. In newly formed 16-cell cysts of primary spermatocytessomewhat away from the apex, the myc-PIWI staining is sharply reduced inthe germline. The staining is only present in the cyst progenitor cells,which are equivalent to follicle cells in the ovary. Once the developingcyst enters the spermatocyte growth phase, myc-PIWI expression iscompletely undetectable. This pattern of expression is maintained in theadult testis which displays the same apical-distal organization but nowcontains more-differentiated post-meiotic germ cells in the basal regionof the testis. Given the role of piwi in testicular germline stem cellmaintenance described herein above, the nuclear localization of PIWI inthe testis is envisioned to be of functional importance.

[0417] PIWI is localized in the nucleoplasm. To further investigate therole of PIWI in the nucleus, the subnuclear localization of the myc-PIWIprotein was examined. In interphase GSCs, myc-PIWI is present insubnuclear areas where DNA staining is the weakest. The myc-PIWIstaining appears as numerous bright foci in DNA-deficient areas that aresometimes connected into a network, complementing the DNA stainingpattern. The DNA-bright area is completely devoid of myc-PIWI. Ininterphase cap cells, myc-PIWI shows a similar staining pattern. Thisstaining pattern suggests that, in interphase somatic and germlinenuclei, myc-PIWI is not associated with the chromatin or the nuclearenvelope, but is localized in the nucleoplasm.

[0418] The nucleoplasmic localization of myc-PIWI is even more evidentduring GSC division. At metaphase, myc-PIWI is not associated withmitotic chromosomes or the nuclear envelope area, but becomes scatteredthroughout the cytoplasm, forming concentrated foci at the cell cortex.The localization pattern remains so throughout anaphase. This dynamiclocalization pattern of myc-PIWI verifies that PIWI is not associatedwith chromosomes or the nuclear envelope, but is a nucleoplasmic factorthat is dispersed into the cytoplasm during mitosis.

[0419] The myc-PIWI protein also shows clear nucleoplasmic localizationin the nuclei of nurse cells, oocytes, and follicle cells in developingegg chambers. In nurse cell nuclei, myc-PIWI is accumulated insubnuclear areas devoid of DNA staining, complementing the DNAlocalization pattern. This complementary relationship is most clearlyillustrated in the oocyte nucleus (germinal vesicle), in which chromatinforms a discrete subnuclear structure called the karyosome that occupiesa spherical area in the nucleus (King, 1970). The myc-PIWI staining isnot detectable in the karyosome, but is conspicuously present in thenucleoplasm that surrounds the karyosome. In follicle cells, myc-PIWI isalso localized in the nucleoplasm in a punctate fashion. Thus, PIWI is anucleoplasmic factor in all types of ovarian cells.

[0420] Therefore, PIWI is localized in the nucleoplasm. In theinterphase germline stem cell nucleus, myc-PIWI mostly accumulates asbright foci in DNA-deficient areas within the nucleus. DNA-bright spotswere observed to be are completely devoid of myc-PIWI. Thiscomplementary staining pattern is also observed in cap cells (CpC).

[0421] In metaphase and anaphase germline stem cells, the myc-PIWIstaining is dispersed throughout the cytoplasm, forming numerous foci atthe cortical region of the cell. In a stage 4 egg chamber (for staging,see King, 1970), myc-PIWI is accumulated in regions of nurse cell nuclei(NCN) and follicle cell nuclei (FCN) devoid of DNA staining. In thegerminal vesicle (GV), the myc-PIWI staining is absent from thekaryosome (K) but present in the nucleoplasm that surrounds it. Theoocyte region of a stage 6 egg chamber showing the complementarymyc-PIWI and DNA staining in the germinal vesicle (GV) and follicle cellnuclei (FCN). A magnified view of a nurse cell nuclei (NCN) in a stage 6egg chamber, showing the largely non-overlapping staining pattern ofmyc-PIWI and DNA.

[0422] PIWI is cell-autonomously required in germline stem cells topromote their division. The above-presented clonal analyses indicatedthat piwi functions in the apical somatic cells of the germarium toregulate GSC maintenance. However, as PIWI is also present in the nucleiof GSCs, the function of PIWI in GSCs was evaluated by removing PIWIfrom a single stem cell using the FLP/FRT-mediated clonal technique (Xuand Rubin, 1993). piwi¹ and piwi² mutations were used for generatingpiwi− deficient clones because they are both strong mutations, asdescribed in Examples 1-4. The piwi⁻ stem cell clones were induced inpiwi FRT/armlacZ FRT transheterozygous flies.

[0423] The amilacZ transgene (Lecuit and Cohen, 1997) is expressed inall somatic and germline cells in the germanium. piwi deficient clonescreated by a mitotic recombination no longer contain the armlacZ geneand can thus be identified by the absence of the lacZ protein. Thecontrol lacZ-negative piwi⁺ germline clones were induced in piwi⁺FRT/armlacZ FRT transheterozygous flies for comparison. Since it takesfour days for a cystoblast to develop into an egg chamber that buds offthe germarium (Margolis and Spradling, (1995)), among piwi-deficientclones only GSC clones can persist over more than five days (FIG. 5B).If no piwi⁻ GSC is observed five days after induced mitoticrecombination, this would clearly indicate the failure of piwi⁻ stemcells to maintain themselves.

[0424] Because recombination events can only occur in mitotically activecells yet all somatic signaling cells are postmitotic, this approachallows the analysis of the cell-autonomous function of piwi in singleGSCs without the complication of generating mutant clones in thesignaling cells. However, even if any somatic clones are induced, theycan be readily identified. Furthermore, by marking individual mutant andwild-type GSCs, the relative division rate and maintenance ability ofeach stem cell can be determined by counting the number of its cystprogeny within the germarium (also see Xie and Spradling, 1998).Finally, the cell-autonomous function of piwi in the germline can beassessed at all stages of oogenesis by examining the development of theindividually marked piwi⁻ germline cysts and egg chambers.

[0425] Mitotic recombination was induced immediately prior to oogenesisand looked foe piwi⁻ GSC clones one week, two weeks, and three weeksfollowing oogenesis. piwi⁻ GSCs were present even three weeks followingoogenesis, consistent with the above-presented observation that PIWI inGSCs is not required for their maintenance.

[0426] The number of marked piwi⁻ and piwi⁺ germline cysts was thenexamined in tester and control germaria to compare the division ratebetween the piwi⁻ and piwi⁺ GSCs within the germarium. For accuratecomparison, germaria that contain only two GSCs, one marked and oneunmarked, were always examined. In wild-type control germaria, markedwild-type stem cells gave rise to approximately 50% of the cysts presentwithin the germarium. This indicated that the lacZ-marked and unmarkedwild-type stem cells divide at the same rate (FIG. 5). However, intester germaria, the number of the marked piwi⁻ germline cysts isconsistently only 25% of marked wild-type cysts (FIG. 5). This analysisreveals that piwi GSCs divided four-fold slower than wild-type GSCs.Thus, in addition to its somatic function, PIWI acts cell-autonomouslyin the stem cells to facilitate their division.

[0427] The piwi⁻ cysts and postgermarial piwi⁻ egg chambers wereexamined by DAPI staining and by Nomarski microscopy for potentialdevelopmental defects. They usually developed normally, suggesting thatpiwi does not play a cell-autonomous role in germline cyst developmentand subsequent stages of oogenesis.

[0428] Therefore, PIWI acts cell-autonomously in GSCs to promote theirdivision. Germaria were labeled with anti-LacZ antibody for piwi⁺ cellsand anti-1B1 antibody for spectrosomes and fusomes. Marked stem cellsand cysts developed from piwi⁻ germline stem cells (GSCS) are recognizedby the absence of lacZ expression. In a piwi⁻ laczFRT/piwi⁺ lacZ⁺ FRTgermanum in the absence of heat shock, no piwi⁻ lacZFRT/piwi⁻ lacZFRTclones were induced. GSCs are distinguished by the presence of thespectrosome (Sp) in direct contact with the terminal filament cells(TFC).

[0429] A piwi⁻ laczFRT/piwi⁺ lacZ⁺ FRT germarium was stained one weekafter heat shock, and showed a wild-type GSC and a mutant GSC, as wellas three piwi⁻ cysts derived from the mutant stem cell. The entireovariole is enveloped by the epithetial sheath (ES), which is differentfrom the inner sheath cells (IS). The graph of FIG. 5 displays therelative division rates of wild-type and piwi⁻ GSCs. The number ofgermaria counted is given in parentheses. Only germaria containing onemarked and one unmarked GSC were examined.

[0430] Overexpression of PIWI increases the number of germline stemcells. Examples 1-4 above establish that the somatic expression of PIWIis essential for GSC maintenance. This somatic induction can be achievedvia a threshold mechanism, in which a certain level of somatic PIWIactivity is required to establish or maintain the stem cell identity,but a higher level of somatic PIWI expression will not increase, thenumber of GSCs. Alternatively, the PIWI-mediated somatic-induction couldact via a dosage-dependent mechanism, in which the somatic level of PIWIdictates the number of GSCs.

[0431] To test these possibilities, PIWI was overexpressed in thesomatic cells of the adult ovary via heat shock induction usinghsp70-Ga1-4 (hsGal4) and ep(2)1024, an EP element inserted in the 5′ UTRof the piwi locus. EP is a modified P element carrying an UAS_(t)promoter oriented to transcribe the flanking genomic sequence upon theactivation of the yeast Gal4 protein (Rorth, (1996)). Specifically,ep(2)1024 in the 5′UTR of piwi is oriented in the same direction of piwitranscription so that it can produce a piwi mRNA containing the completePIWI open reading frame. The UAS_(t) promoter is active in the soma butnot in the germline (Rorth, 1998), and its somatic specificity wasconfirmed by examining the ovarian expression of the UAS_(t)-GFPreporter gene under experimental conditions. Because endogenous PIWI isexpressed in all somatic cells in the germarium, by constructing aDrosophila strain that contains hsGal4 and ep(2)1024, it was possible toover-express, but not ectopically express, piwi specifically in thesomatic cells by heat shock induction.

[0432] To verify that ep(2)1024 can express a functional PIWI protein,ep(2)1024 was overexpressed by heat shocking hsGal4 in anep(2)10241/piwi¹ mutant background. Because ep(2)1024 is a strongloss-of-function mutation of piwi, ep(2)1024/piwi¹; hsGal4 females andmales in the absence of heat shock display typical piwi phenotype. Incontrast, the same mutants, after receiving daily heat shock treatmentfrom the second instar larvae to three days after eclosion, producedsignificantly restored fertility in both ep(2)1024/piwi¹ females andmales. The morphology of both the ovaioles and testes in the heatshockedmutants are indistinguishable from their wild-type siblings. Inaddition, the fertility of the rescued mutants is comparable to theirwild-type siblings. Heatshock starting at the pupal stage does notrescue the fertility. Therefore, ep(2)1024 can produce a functional PIWIprotein capable of rescuing the piwi mutant phenotype when expressed insomatic cells.

[0433] ep(2)1024 was then used to overexpress PIWI in wild-type adultfemales, and its effects on GSCs were analyzed. The females wereheat-shocked at a 12-hour interval for three days and then dissected.The ovaries were stained with anti-VASA antibody (Hay et al., (1990)) tohighlight germ cells and anti-1B1 antibody (Zaccai and Lipshitz, (1996))to outline somatic cells as well as to label spectrosomes and fusomesthat mark individual stages of germline development in the germarium(Lin and Spradling, (1995)). Wild-type germaria typically have 2-3spectrosome-containing cells, with one often being a cystoblast that isnot associated with the terminal filament. This is also the case foreuninduced ep(2)1024/+ germaria. Interestingly, the number of thespectrosome-containing germ cells increased to an average of 7.5 cellsper ep(2)1024/+ germarium following heat shock induction. In the mostextreme case, up to 15 spectrosome-containing cells were observed in asingle germarium. This increase is not due to the expression of hsGal4alone, since ovaries from hsGal4 females subjected to the same heatshock regime were indistinguishable from wild-type. Thus, overexpressionof PIWI in the soma leads to a 34 fold increase in the number of GSCsand/or cystoblasts.

[0434] Thus, the observation that EP(2)1024 expression rescues the piwi¹mutant phenotype was shown via DAPI staining of the gonads fromep(2)1024/piwi¹; hsGAL4 females and males either without or with heatshock. In the absence of the heat shock, the ovaries and testes displaythe typical piwi¹ mutant phenotype. Following hsGal4-inducedover-expression of the ep(2)1024 in the soma, significant rescue of themutant phenotype was observed in both females and males. Specifically;in females, germline stem cells are maintained and a string ofdeveloping egg chambers was observed. Similarly, in males, germline stemcells were also rescued, and spemmatogenesis was fully restored.

[0435] To distinguish whether the spectrosome-containing cells are stemcells or cystoblasts, the germaria were stained with anti-cytoplasmicBAM (BAM-C) antibody, since-BAM-C is only expressed in cystoblasts andearly mitotic cysts but not in GSCs (McKearin and Ohlstein, (1995)). Inpiwi-overexpressed germaria, BAM-C staining is strongly present in earlycysts. However, BAM-C staining is conspicuously absent from all thespectrosome-containing cells. This observation suggests that theectopically induced spectrosome-containing cells are GSC-like cells.

[0436] The observation that somatic PIWI over-expression increases thenumber of germline stem cells was supported a confocal z-series analysis(10 μm stacks) of wild-type germarium immunofluorescently labeled withanti-1B1 antibody. At the apical tip of region 1 in direct contact withthe terminal filament, two germline stem cells containing spectrosomes(Sp) were observed. Posterior to germline stem cells, developinggermline cysts containing branched fusomes (Fu) were observed. Agermarium from a ep(2)1024; hsGal4 female was observed to display manygermline stem-like cells following 4 days of heat shock treatment, with12 shown in this focal plan as indicated by spectrosomes (Sp; stainedwith anti-1B1 antibody) and the absence of the BAM-C protein. Thestem-like cells were evenly distributed between regions 1 and 2a. TheBAM-C protein was only present in a cyst containing a branched fusome inregion 2a.

[0437] A germarium containing ectopic stem-like cells was stained withanti-BrdU and anti-a-spectrin antibodies following BrdU incorporation asdescribed above in the Materials and Methods. Some mitotically activegermline cells which have incorporated BrdU in their nuclei wereobserved. A fusome-containing germline cyst (Fu) was present in region 1but not in contact with somatic cells. A germarium from an ep(2)1024;hsGal4 female subjected to 4 days of heat shock treatments followed by4-5 days of culture at room temperature was also stained. The stainingsindicated anti-VASA and anti-1B1 antibody signals, respectively. Thisgermarium, like a typical wild-type germarium, contained two germlinestem cells contacting the apical somatic cells and a cystoblast that isone cell away from the terminal filament. In addition, the number offusome-containing cysts (Fu) have increased to a normal level.

[0438]FIG. 10 depicts a graphical comparison of the number ofspectrosomes present per wild-type germarium versus perpiwi-overexpressed germarium. The number of germaria examined (n) is inparentheses. All germaria are oriented with anterior toward the left.

[0439] The ectopic GSC-like cells appear to be functional GSCs. First,they incorporate BrdU, an indicator of DNA replication, at a levelsimilar to the wild-type GSCs. This suggests that they are not arrestedin the cell cycle. Second, these GSC-like cells can all differentiatewithin four days following the withdrawal of heat shock; leaving thegermaria with only 2-3 GSCs at their normal locale. No signs of celldeath, such as pycnotic nuclei or apoptotic bodies, were detected byDAPI staining and Nomarski optics. This suggests that the GSC-like cellsare capable of oogenesis. Thus, the dependence of GSC number on the PIWIlevel reveals that piwi-mediated somatic signaling controls the numberof GSCs via a dosage dependent mechanism.

[0440] The ectopically induced GSCs show an even distribution patternalong the germarial axis. In wild-type germaria, spectrosome-containingcells are exclusively located in the apical area of region 1, either indirect contact with or one-cell away from the terminal filament (forstem cells and cystoblasts, respectively). However, following PIWIoverexpression, spectrosome-containing cells were evenly expanded alongthe somatic germarial wall to both regions 1 and 2a. All thespectrosome-containing cells are in apparent contact with the somaticcells; there is no apparent gradient of the stem-like cell distributiontowards the terminal filament. In wild-type germaria, inner sheath cellsexpress as much PIWI as cap cells, and much more than the main terminalfilament cells, yet there are no GSCs located near them in posteriorregion 1 or region 2a. Therefore, the appearance of the ectopicstem-like cells in the entire regions 1 and 2a followingpiwi-overexpression suggests that a very high level of PIWI in the innersheath cells expands the stem cell domain to region 2.

[0441] Over-expression of PIWI increases the rate of germline stem celldivision. Since over-expression of piwi in somatic cells increases thenumber of GSCs, yet loss of piwi function from the soma and germline inpiwi mutants abolishes stem cell division and thus their maintenance,one might expect that over-expression of piwi in the soma may alsoincrease the rate of GSC division. To address this possibility, themitotic index of GSCs in piwi-overexpressed females was directlyquantified. GSC division is unique in that it has an unusually longtelophase-to-interphase period characterized by the presence of a dumbbell-shaped telophase spectrosome spanning the two daughter cells. Dengand Lin, (1997); de Cuevas and Spradling, (1998). The morphologicallydistinctive telophase spectrosome provides an easy marker for theaccurate measurement of the telophase/early interphase index, which is adirect quantitation of the frequency of GSC division.

[0442] To avoid a bias in quantifying the division frequency due to thepresence of an increased number of GSC-like cells in piwi-overexpressedgermaria, the 2-3 anterior-most GSCs that contact the terminal filamentwere quantified, and the same number of stem cells in the wild-type anduninduced ep(2)1024 controls were quantified. In the wild-type anduninduced ep(2)1024 controls, 20-25% of germaria contained a single GSCwith a telophase spectrosome; only 2-3% of germaria contained two GSCswith telophase spectrosomes. These frequencies are increased to 31% and13% in piwi-overexpressed germaria. Moreover, 0.3% of thepiwi-overexpressed germaria contain three GSCs with a telophasespectrosome, a situation that was not observed in the control germaria.These results indicate that in addition to an increase in the number ofstem cells, piwi overexpression also increases the rate of GSC division.Because the GSCs under examination are located in their normal niche,the increase in their mitotic frequency should be mostly, if not all,due to the increased PIWI expression in the terminal filament.

[0443] Four micrographic panels of anti-1B1 images of germariacontaining 0, 1, 2, and 3 telophase spectrosome-containing germline stemcells were prepared and examined. The frequency of observing suchgermaria in the wild-type control, the uninduced ep(2)1024 control, andthe piwi-overexpressed germaria is presented in Table 5. The number ofgermaria counted for each genotype (n) is given in parentheses. TABLE 5Percentage (%) of GSC in Telophase Telophase W.T. EP (−) HS EP (+) HSGSCs (n = 100) (n = 100) (n = 100) 0 73 77 53 1 25 20 31 2 2 3 13 3 0 00

Discussion

[0444] Stem cells are characterized by two common properties: thecapacity for self-renewal and the ability to propagate numerous progenyfated for further differentiation (for review, see Morrison et al.,1997; Lin, 1997). The unique self-renewing division of stem cells iscontrolled by both intracellular and extrinsic signaling mechanisms(Knoblich, 1997; Lin, 1997). Example 5 shows that piwi encodes a novelnucleoplasmic protein present in both somatic and germline cells of bothsexes. Moreover, the single stem cell clonal analyses and piwioverexpression experiments establish the important function of piwi inboth the intracellular and the cell-cell signaling mechanisms. Example 5thus reveals a novel nuclear factor whose activity modulates the numberof GSCs and the rate of their division.

[0445] The role of PIWI as a nucleoplasmic factor in somatic signaling.The immunofluorescence analysis of the fully functional-myc-PIWI proteinreveals that PIWI is a nucleoplasmic protein. Consistent with RNA insitu analyses, PIWI is present in both the soma and the germline duringoogenesis. In Example 5 the cell-autonomous function of PIWI as amitotic promoter in GSCs is revealed. PIWI is localized in thenucleoplasm in both the apical somatic cells and GSCs. Hence, thenucleoplasm is the likely site of PIWI function, and in accordance withone aspect of the present invention, PIWI is the first nucleoplasmicfactor known to play a key role in controlling GSC division.

[0446] As a nucleoplasmic factor in the apical somatic cells, PIWI isexpressed in three types of post-mitotic cells that form a somatic capsurrounding GSCs and their immediate daughter cells: the main terminalfilament cells, the cap cells, and the inner sheath cells. Among thesecell types, the main terminal filament cells express a very low level ofPIWI while cap cells and inner sheath cells express a very high level ofPIWI. Since cap cells are in direct contact with GSCs, it is envisionedthat PIWI expression in cap cells plays a role in somatic signaling.

[0447] Since PIWI is a nucleoplasmic protein, it is unlikely a somaticsignal itself, but rather an essential component of the somaticsignaling machinery responsible for producing the signal. Given itsnucleoplasmic localization, PIWI might be involved inpost-transcriptional mRNA processing in the nucleus or in nuclearfunctions indirectly related to gene expression. In either case, thesomatic activity of PIWI acts via a dosage dependent mechanism tocontrol the number of GSCs in the germline. Therefore, piwi can help todefine a stem cell niche in the germarium for GSC maintenance, with thesize of the niche corresponding to the level of PIWI activity.

[0448] A number of genes, including fs(1)Yb (King and Lin, 1999) and dpp(Xie and Spradling, 1998), have recently been shown to play roles in thecell-cell signaling mechanism in maintaining GSCs. Among these genes,dpp is the only known signaling molecule. DPP over-expressiondramatically expands the number of GSCs and prevents theirdifferentiation, leading to tumorous germaria filled withundifferentiated GSC-like cells (Xie and Spradling, 1998). Thisoverexpression phenotype, in sharp contrast to that of piwi, suggeststhat DPP is not the downstream signal of the piwi-mediated mechanism,even though it may be one of the signals. fs(1)Yb, on the other hand,shares a very similar loss-of-function phenotype with piwi. These twogenes are more likely to be involved in the same somatic signalingpathway.

[0449] The role of PIWI as a nucleoplasmic factor in germline stemcells. PIWI is expressed dynamically in the germline of the germarium.In region 1, PIWI is present in the nucleus of GSCs at a high level butis sharply down regulated in cystoblasts and early mitotic cysts. Thisexpression pattern correlates well with the mitosis-promoting functionof PIWI in GSCs as revealed by single stem cell clonal analysis. Thedifferential distribution of proteins between the stem cell and thecystoblast has also been observed for several other factors known to beimportant for GSC division or differentiation. For example, PUM isrequired for GSC maintenance (Lin and Spradling, 1997; Forbes andLehmann, 1998) and is accumulated at a high level in GSCs but a lowlevel in the cystoblasts (Forbes and Lehmann, 1998; Parisi and Lin,1999). On the other hand, NOS and BAM-C are required for cystoblastdevelopment and are localized either preferentially or exclusively inthe cystoblast.

[0450] All of these molecules, except for BAM-C, are known to playimportant roles in other developmental processes (Cox et al., 1998;Parisi and Lin, 1999). For example, the germline expression of PIWIduring oogenesis also provides. PIWI as a maternal component essentialfor embryogenesis (Cox et al., 1998). Therefore, the asymmetric fatesbetween GSCs and cystoblasts might not be determined by unique “stemcell factors” or “cystoblast factors”. It, instead, may be created byunique combinations of various regulatory molecules that are repeatedlyused throughout development.

[0451] This pleiotropic function of stem cell genes is also manifestedwithin the stem cell mechanisms, in which piwi is involved in bothsomatic signaling and in cell-autonomous mitotic functions. piwi thusacts in two distinct cell types and in two different mechanisms.Although applicant does not desire to be bound by any particularmechanism of action, it is contemplated that the PIWI protein directlyor indirectly mediates gene expression in the nucleus either at thetranscriptional or post-transcriptional level. The different piwi targetgenes or their products in different cell-types then lead to thedistinctively different cellular function of piwi.

Example 6 PIWI is a Pole Plasm Component Required for Pole CellFormation, Migration and Transcriptional Quiescence

[0452] This Example shows that PIWI is also a maternally deposited polargranule component that plays a key role in pole cell formation,migration, and transcriptional quiescence. However, unlike most othergenes known to be required for pole cell formation, piwi is specificallyrequired for pole cell formation with no effect on abdominal patterning.PIWI is dynamically expressed during germ plasm assembly and pole cellformation. The protein is first expressed in polar granules at theposterior end of the embryo and is subsequently expressed in the nucleiof all primordial germ cells throughout embryogenesis. Furthermore, thepiwi gene product directs germ plasm assembly and controls the number ofpole cells formed at the posterior pole of the Drosophila embryo. Theseresults indicate that piwi has many of the properties of a germcell-specific determinant.

Materials and Methods

[0453]Drosophila strains and culture. All strains were grown at 25° C.on yeast-containing molasses/agar medium. The following fly strains wereused in this study: w; piwi¹, FRT^(40A)/CyO and w; piwi², FRT^(40A)/CyO(as disclosed in Examples 1-4) were used to generate maternal clones; y,w P[hsFLP]¹²; P[ovo D1]^(2L), FRT^(40A)/CyO (Chou and Perrimon, 1996);P[myc-PIWI] insertions on the second and X chromosomes were used toincrease the piwi copy number as well as to visualize PIWI proteinexpression; 4× oskar flies bear two wild-type copies of oskar on thethird chromosome and two transgenic rescue copies of oskar on the secondchromosome (Ephrussi and Lehmann, 1992. OregonR (Ore-R) serves as thewild-type control in all experiments.

[0454] Immunohistochemistry. Ovaries were dissected, fixed and stainedas described in Lin et al., 1994. Embryos were collected, fixed andstained according procedure described by Patel etal. (1989). Forimmunofluorescence staining the following antisera were used: polyclonalanti-VASA antibody (1:2000; Hay et al., 1990); monoclonal anti-MYCepitope antibody (1:50; Evan et al., 1985); monoclonal anti-SXL antibody(1:500; Bopp et al, 1991). All the fluorescence-conjugated secondaryantibodies were from Jackson Immunoresearch Laboratory, Westgrove, Pa.and were used at 1:200 dilution. Immunofluorescently labeled sampleswere also counterstained with DAPI as described previously (Lin andSpradling, 1993). Micrographs were taken using either a Zeiss AXIOPLAN®microscope or a Zeiss LSM410™ confocal microscope (Zeiss, Oberkochen,Germany) as described herein above.

[0455] Maternal clones. To generate embryos maternally depleted for piwithe following fly strains were crossed: y, wP[hsFLP]¹²; P[ovo^(D1])^(2L), FRT^(40A)/CyO males were crossed to w, piwi¹, FRT^(40A)/CyOvirgin females to produce y,w P[hsFLP]¹²; P[ovoD¹]^(2L),FRT^(40A)/piwi¹, FRT^(40A) progeny. Identical crosses were performedwith w; piwi², FRT^(40A)/CyO virgin females. Crosses were carried outfor two days to produce transheterozygous progeny. After 2 days, adultswere transferred to fresh vials. Larvae were heat shocked on days 3 and4 for one hour in a 37° C. water bath to induce mitotic recombination.The heat-shocked females with germline clones were crossed to Ore-Rmales or to piwi² mutant males. Embryos collected from these femaleswere subsequently analyzed by immunofluorescence microscopy. Due to thepresence of a copy of the ovo^(D1) gene in these females, only piwi⁻germline clones can give rise to a mature egg.

[0456] Pole cell formation is dosage-dependent for piwi. Single copyembryos are from piwi¹/CyO females and two copy embryos for both piwiand oskarare from Ore-R females. For the four-copy piwi embryos, aP[myc-PIWI] rescue transgene was homozygoused on a wild-type secondchromosome; two such lines were examined for their effects on embryonicdevelopment. For the four-copy osk embryos, an osk rescue transgene washomozygoused on the second chromosome of an otherwise wild-type fly asdescribed by Ephrussi and Lehmann, 1992. For the six-copy piwi embryos,P[myc-PIWI] transgenes on the X chromosome and second chromosome werehomozygoused to generate four transgenic copies in addition to the twowild-type copies on the second chromosome. For maternally depletedembryos, germline clones were produced as described above. Embryos fromfemales carrying germline clones were collected and analyzed to examinethe role of piwi in germline development. For pole cell counts, allembryos were stained with anti-VASA antibody and the pole cells of 50embryos of each maternal genotype, described above, were counted intriplicate for each embryo. The pole cell count reflects the initialnumber of pole cells present shortly after budding at the posterior poleand prior to any amplification or migration. Embryo staging wasperformed as described in Zalokar and Erk (1976) according to the numberof nuclei present.

Results

[0457] PIWI encodes a polar granule component with a dynamic behaviorduring pole cell formation. As disclosed above, piwi is maternallyrequired for embryogenesis. To examine piwi function in embryogenesis,PIWI protein expression in embryos was analyzed using a myc-tagged piwitransgene. Embryos were collected from females carrying a P[myc-PIWI]transgene and subsequently stained these embryos with antibodies againstMYC to visualize P[myc-PIWI] protein expression and VASA, a polargranule component and pole cell-specific marker. Embryos werecounterstained with DAPI to visualize nuclei and allow a stage-specificanalysis of PIWI expression during embryogenesis (Zalokar and Erk,1976).

[0458] PIWI is first expressed in a crescent at the posterior pole ofthe embryo during mitotic cycles 1-7, where it is co-localized with VASAduring these cycles, including localization to polar granules of whichVASA is a component. Thus, piwi encodes a polar granule component.During mitotic cycles 8-10, PIWI is dynamically expressed in the formingpole cells. At this stage in embryogenesis, nuclei have migrated fromthe center of the embryo and some of them enter the posterior poleplasm. These posterior nuclei become the first to cellularize as theybud off from the embryo to form pole cells. At the onset of pole cellformation, during mitotic cycles 8 and 9, PIWI is expressedcytoplasmically in an overlapping pattern with VASA in polar buds.However, at mitotic cycle 10 when pole cell formation and budding iscomplete PIWI completely translocates to the nucleus of all pole cells.PIWI is also zygotically expressed in somatic cells at the stage. PIWIremains nuclear in pole cells throughout germ cell migration and finallygonad coalescence.

[0459] Maternal PIWI is required for pole cell formation. To analyze thepotential requirement of piwi in embryogenesis embryos maternallydepleted for piwi were generated. Females with piwi¹ germline stem cellclones were crossed to wild-type males and embryos from thesefemales-were collected. To assay germline formation and development,germ cells were visualized by staining with an anti-VASA antibody. Inthe absence of maternal piwi product, a dramatic reduction in the numberof pole cells formed was observed. Maternally depleted piwi embryoscontain on average 7.5±5 pole cells at mitotic cycle, 10, the pole cellformation stage, in contrast to the 24±5 pole cells formed in wild-typeembryos at the same stage. These results indicate that maternal piwi isrequired for pole cell formation as a wild-type paternal copy of piwi⁺isinsufficient to rescue the pole cell formation defect.

[0460] Following pole cell formation there are typically one to twoamplificatory mitoses in the pole cell population such that 30-40 polecells are present prior to germ cell: migration. There is no additionalamplification of the pole cell population once germ cell migration hascommenced (Williamson and Lehmann, 1996). To determine whether piwi isrequired for the amplification of the pole cell population, embryoslacking maternal piwi for the number of pole cells present before germcell migration and after gonad coalescence were examined. There is noamplification of germ cells in the absence of maternal piwi; rather, asmall decrease in the average number of germ cells present at gonadcoalescence was observed. Prior to germ cell migration piwi⁻ embryoscontained 7.5±5 germ cells, however at germline coalescence the averagenumber of germ cells dropped to 4.5±3 germ cells. Thus, in addition toits requirement for pole cell formation, maternally deposited piwi alsoplays a role pole cell amplification and survival.

[0461] The maternal effect of piwi on pole cell formation is similar tothat observed with the posterior group genes which are also required forpole plasm assembly and pole cell formation. To examine whethermaternally depleted piwi embryos have any abdominal defects, Nomarskioptics were used to examine the segmentation pattern of these embryos.There is no readily apparent defect in abdominal patterning. Thus, incontrast to the posterior group genes, piwi does not appear to berequired for proper abdominal patterning of the embryo.

[0462] Maternal PIWI is required for Dole cell migration. To analyze theeffect of removing maternal piwi function on germ cell migration,embryos from females bearing piwi mutant germline stem cell clones werecollected and stained for the germ cell specific marker VASA. Inaddition to its role for proper pole cell formation and proliferation,piwi activity also plays a role in successful germ cell migration.During pole cell migration in wild-type embryos, germ cells exit theposterior migration and then actively migrate through the endoderm alongits basal surface until making contact with the overlying mesodermalcells (Warrior, 1994). At late stage 10 to early stage 11 (for staging,see Zalokar and Erk, 1976), germ cells transfer from the endoderm intothe mesoderm where they will associate with somatic gonadal precursorcells which give rise to the gonadal mesoderm. Finally, at stage. 14 ofembryogenesis, germ cells coalesce with somatic gonadal precursors inparasegment 10 to establish the primordial gonad.

[0463] However, in embryos lacking maternal PIWI, defects in germ cellmigration are seen from stage 10 onwards which marks the active phase ofgerm cell migration. As germ cells exit from the posterior midgutpocket, they fail to undergo directional migration over the surface ofthe endoderm. Rather, they are found migrating in a random fashion tomany different sites within the embryo. In some cases highly aberrantgerm cell migration into the anterior end of the embryo was observed.The random patterns of germ cell migration in these mutant embryosindicates a requirement for maternal PIWI in germ cell guidance. Inaddition to the defect in germ cell migration, defects in overall polecell morphology were observed. In contrast to wild-type pole cells, polecells maternally depleted of piwi are of variable size and shape anddisplay unusual blebbing and aberrant pseudopodia. This indicates thatmaternal piwi also plays a role in proper pole cell morphology whichmight be essential for proper pole cell migration.

[0464] Previously, it has been demonstrated that signals produced bysurrounding somatic tissues act to guide germ cells by both attractiveand repulsive forces (Jaglarz and Howard, 1994; 1995; Zhang et al.,1997; Van Doren et al., 1998). However, few genes have been identifiedthat control germ cell migration and are expressed in germ cellsthemselves. The expression of piwi in pole cells during migrationsuggests that piwi function is cell-autonomous. This is similar to nanosand pgc-1 both of which are expressed in germ cells and are required fornormal germ cell migration (Forbes and Lehmann, 1998; Kobayashi et al.,1996). These data indicate that germ cell migration is controlled byboth instructive signaling mechanisms as well as by cell-autonomousmechanisms within migrating germ cells.

[0465] Maternal PIWI plays a role in the transcriptional Quiescence ofDole cells. Transcriptional repression in germ cells has been implicatedas a key mechanism in the establishment and maintenance of the germ cellfate (for review, see Seydoux and Strome, 1999). In Drosophila zygotictranscription in the soma is detectable as early as one hour after egglaying (AEL; Pritchard and Schubiger, 1996). In the germline, however,general RNA synthesis is not detected until 3.5 hours AEL at embryonicstage 8 (Zalokar, 1976) and poly (A) containing mRNA transcripts are notobserved in early germ cell nuclei (Lamb and Laird, 1976). Few geneshave been shown to be specifically transcribed in pole cells and amongthe known genes, vasa is the earliest gene at embryonic stage 9-10 (Hayet al., 1988b). Transcription of all other genes to be expressed in polecells is not observed until the primordial gonad reinitiatesproliferation at embryonic stage 16-18 (Mevel-Ninio et al., 1995).Transcription is reinitiated in pole cells just before they migratethrough the midgut epithelium approximately 3.5 hours AEL (Zalokar,1976).

[0466] To analyze the potential role of piwi in the regulation oftranscriptional quiescence in early germ cells, embryos maternallydepleted of piwi were double-labeled with VASA, a germ cell specificmarker, and SXL, a somatic sex-specific marker which is normally absentin pole cells. Ectopic expression of SXL protein in pole cells prior toembryonic stage 9 was observed. This indicates that the mechanism whichnormally maintains transcriptional quiescence in pole cells is dependenton proper piwi function.

[0467] piwi Dose Determines the Initial Number of Pole Cells. Given thecritical role of piwi in pole cell formation and development, the roleof piwi in germ plasm assembly was further evaluated by examining therelationship between piwi gene dosage and the number of pole cellsformed, which is also a reflection of the amount of pole plasm formed atthe posterior end of the egg. The piwi copy number was increased infemales from one to four and collected embryos from these females toexamine the number of pole cells formed. In identically staged samples,embryos with one or two copies of the piwi gene contained on average24±5 pole cells. However, a two-fold increase in piwi gene dosageoverwild-type levels yields embryos containing on average 50±7 polecells at the same developmental stage. This phenotype is strikinglysimilar to the phenotype displayed when dskargene dosage is increasedfrom one to four copies (Ephrussi and Lehmann, 1992). Like oskar, atwo-fold increase in piwi gene dosage leads to an increase in both theamounts of known pole plasm components, such as VASA, at the posteriorpole of the embryo and the number of pole cells formed. However, incontrast to oskar, increasing piwi gene dosage does riot cause defectsin abdominal patterning or polarity (Ephrussi and Lehmann, 1992). Insupport of the apparent linear effect of piwi gene dosage on the numberof pole cells formed, we found that a three-fold increase in piwi genedosage results in embryos containing on average 66±7.5 pole cells,representing a three-fold increase over the number of pole cells inwild-type embryos at the same developmental stage.

Discussion

[0468] piwi plays a role in embryonic germline determination. piwiencodes a polar granule component which is maternally required for polecell formation. However, unlike other posterior group genes which arealso required for abdominal patterning, piwi is specifically involved inpole cell formation. This Example indicates that, unlike the posteriorgroup genes, piwi does not appear to be required for the properlocalization of the abdominal determinant, nanos.

[0469] At the molecular level, germ plasm assembly occurs in a stepwiseordered fashion. The presence of the VASA protein in the smallpopulation of pole cells formed in embryos lacking maternal piwiindicates that piwi must act downstream of vasa and therefore oskar inpole cell formation (for review, see Rongo and Lehmann, 1996). In thishierarchy of gene activity, oskar is required for the localization ofvasa and tudor to the pole plasm. In addition, all three gene productsare polar granule components which like piwi are required for pole cellformation. However, piwi has a role in pole cell formation which isdownstream of the requirements for germ plasm assembly. This requirementis very similar to that reported for other putative germ celldeterminants including, germ cell-less (gcl) (Jongens et al., 1992),polar granule component-I (pgc-1) (Nakamura et al., 1996), and mtlr RNA(Kobayashi et al., 1993). All of these genes act downstream of tud andare required for pole cell formation, but not for abdominalsegmentation. These results suggest piwi can act as a maternallydeposited germ cell determinant downstream of tud as well.

[0470] Maternal PIWI level determines the initial number of pole cells.A direct relationship was observed between the dosage of the piwi geneand the concentration of other pole plasm components, such as VASA, atthe posterior pole of the embryo. This phenotype is highly similar tothat observed when oskar copy number is increased in embryos (Ephrussiand Lehmann, 1992). For both piwi and oskar, an increase in gene copynumber leads to an accumulation of pole plasm components which isindependent of the dosage of other component genes. Therefore, both piwiand oskar can modulate the number of pole cells formed through a genedosage effect via regulating the amount of pole plasm. Pole cellformation involves a hierarchy of gene expression. It has been proposedthat oskar acts as a limiting factor for all aspects of pole plasmfunction (Ephrussi and Lehmann, 1992). The highly similar phenotypesobserved in embryos carrying four copies of either piwi or oskar,suggest that osk is not the only limiting factor for germ cell formationand that piwi might be a downstream target of oskar in determining polecell formation. The presence of VASA protein in germ cells of embryosmaternally depleted for piwi also suggests that piwi is requireddownstream of oskar for pole cell formation (Ephrussi and Lehmann,1992).

[0471] As disclosed herein, piwi is a member of a large class ofevolutionarily conserved genes. Intriguingly, piwi is related toaubergine, a Drosophila gene required to promote oskar translation(Wilson et al., 1996). piwi, however, is not likely to promote oskartranslation since piwi-depleted embryos do not show osk-like defects.Alternatively, piwi may act downstream of oskar, but in a completelyoskar-independent fashion.

[0472] Previous analyses have revealed that pole plasm assembly is atwo-step process beginning in oogenesis. The first step is thelocalization of oskar mRNA to the posterior pole of the oocyte. Thesecond step is the recruitment of vasa and tudor by oskar. Both vasa andtudor are required for pole plasm assembly; Downstream of tudor there isa bifurcation of the hierarchy leading to germ cell determination andthe proper localization of the abdominal determinant nanos,respectively. In the germ cell determination branch are potential germcell determinants which are required for pole plasm assembly and, polecell formation, but are not required for abdominal patterning. The datapresented in the Examples suggests that piwi is likely to act downstreamof oskar in the germ cell determination branch as a germ celldeterminant.

[0473] piwi's potential role as a germ cell determinant can be furtheraddressed by ectopically expressing the piwi mRNA at the anterior end ofthe embryo to examine whether it can induce pole cell formation. Thisapproach has used in identifying molecules crucial to germ celldetermination (Ephrussi and Lehmann, 1992; Jongens et al., 1994).

[0474] Maternal PIWI is required for migration and transcriptionalquiescence of germ cells. In addition to initial pole cell formation,piwi activity is required for successful germ cell migration. MaternalPIWI is nuclearly localized to all pole cells as they form and remainsnuclear throughout germ cell migration and primordial gonad formation.In the absence of maternal PIWI, germ cells migrate in a highlyrandomized fashion with few germ cells actually reaching the gonadalmesoderm. Recent studies have identified several genes which regulategerm cell migration (Moore et al., 1998; Zhang et al., 1996), however,unlike piwi, all of these genes appear to be required in the soma andnone of these mutants result in such randomized migration of germ cellsas that observed in piwi mutant germ cells. In addition to piwi,maternal NOS has also been shown to act cell-autonomously in germ cellsto facilitate their migration (Forbes and Lehmann, 1998). Somaticsignaling has been shown to exert both attractive and repulsive forceson germ cells in order to guide their migration (Zhang et al., 1996; VanDoren et al., 1998a). The aberrant germ cell migration observed inembryos lacking maternal PIWI could result from a failure to respond tothese somatic guidance cues perhaps due to the absence of specificreceptors for the cues on the surface of germ cells. In any case, theseresults indicate that soma-germline signaling is essential for propergerm cell migration and that maternally supplied products, such as PIWIand NOS, are required for successful germ cell migration.

[0475] The small decrease in the average number of germ cells aftermigration was initiated in piwi-depleted embryos indicates that maternalPIWI also plays a role in germ cell survival. Both in vitro and in vivostudies suggest that the mammalian stem cell genes White (W) and Steel(So) also function in the survival of primordial germ cells (Gomperts etal., 1994). SI mutants are sterile due to poor germ cell survival andimproper migration (Witte, 1990). Therefore, PIWI and SL play a similarrole in the proliferation and migration of primordial germ cells.However, the SL function is zygotically required, just as nos and ovo inDrosophila piwi is the first clearly demonstrated case where amaternally provided product functions in the survival of pole cellsduring embryogenesis.

[0476] This Example further reveal that maternal PIWI is required fortranscriptional repression in early germ cells. Transcriptionalrepression has been implicated as an essential mechanism in theestablishment and maintenance of the germ cell fate (for review, seeSeydoux and Strome, 1999). Previous studies indicate that the repressionof RNAP II activity in early germ cells may be part of an evolutionarilyconserved mechanism that distinguishes the germline from the soma duringearly embryogenesis (Seydoux and Dunn, 1997). Germ cells in bothDrosophila melanogaster and C. elegans have been shown to lack aspecific phosphoepitope of RNAP II until germ cells associate with thegut primordium during gastrulation (Seydoux and Dunn, 1997). Thephosphorylation of RNAP II is a prerequisite for its transcriptionalactivity. Thus, the absence of the phosphoepitope suggests that RNAP IIactivity is inhibited in early embryonic germ cells (Seydoux and Dunn,1997; Van Doren et al., 1998).

[0477] While applicant does not wish to be bound by a particularmechanism of action, piwi could function to repress transcription inearly germ cells by regulating the expression of this specificphosphoepitope of RNAP II. In C. elegans, the pie-1 gene product hasbeen shown to function in transcriptional repression of the germ celllineage (Mello et al., 1992). In Drosophila, piwi represents the firstgene implicated in regulating transcriptional quiescence in germ cells.Prior to zygotic gene expression in the Drosophila embryo, germ cellsundergo a wide range of morphological and cellular behaviors includingprecocious cellularization, translational regulation (Dalby and Glover,1993), germ cell-specific cell cycle regulation (Sonnenblick, 1941) aswell as the development of a migratory cellular morphology (Jaglarz andHoward, 1995).

[0478] Therefore, the germ plasm, which is maternally provided, mustcontain a combination of factors which controls these processes in earlygerm cells. Maternally provided factors in the germ plasm must functionto repress germ cell gene expression as well as to activate specificgene expression in primordial germ cells at the appropriate time. Thus,the mechanisms for timing the onset of zygotic gene expression in thegermline should be maternally provided as cell-autonomous factors togerm cells. PIWI is envisioned to represent such a factor to maintaintranscriptional quiescence until the appropriate developmentaltimepoint. The loss of transcriptional repression in germ cells lackingmaternal PIWI could result in the loss of normal germ cell propertiesdue to differentiation, which could account for the defective pole cellmigration.

[0479] PIWI as a factor for maintaining totipotentcy in germ cells.Primordial germ cells are the only population of cells in the organismthat retain totipotent potential. Based on studies of genes such aspie-1, transcriptional repression has emerged as a key mechanism thatprevents germ cells from expressing genes that would cause them to enterother lineages and differentiate (Mello et al., 1992; for review, seeSeydoux and Strome, 1999). piwi is essential for the maintenance andproliferation of germline stem cells in both males and females. Germlinestem cells, like primordial germ cells, should be pluripotent; themaintenance of their identity is vital for the propagation of thespecies. Furthermore, PIWI is specifically expressed in all pole cellnuclei and is required for proper germ cell formation andtranscriptional repression. Particularly, piwi gene dosage is directlyrelated to the number of pole cells formed. These data, taken together,suggest that piwi has many of the essential properties required forestablishing and maintaining pluripotency during germline development.

Example 7

[0480] miwi Contributes to the Long Term Self-Renewing Ability ofHematopoietic Stem Cells

[0481] To test for the role of miwi in long term self-renewing abilityof hematopoietic stem cells (HSCs), miwi^(+/−) (marked by ly5.1) andmiwi^(−/−) (marked by 5.2) bone marrow cells were co-transplanted intoirradiated ly5.1 mice at ratios of 1:1 and 2:1, respectively. Thepercentage of hematopoietic cells was assayed at 2, 6, and 12 weeksfollowing bone marrow transplantation. In both co-transplantationexperiments, the percentage of hematopoietic cells derived frommiwi^(−/−) bone marrow steadily decreased over the course of the assay(FIG. 14). Particularly, in the co-transplantation experiment using a2:1 ratio of miwi^(+/−) to miwi^(−/−), miwi^(−/−) hematopoietic cellswere completely depleted by 12 weeks following co-transplantation. Theseresults suggest that miwi contributes to the long-term self-renewingability of HSCs.

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1 21 1 3047 DNA Drosophila sp. CDS (84)..(2612) misc_feature(120)..(120) n = a or c, Xaa = Leu or Ile 1 ctgagtccaa agcgtcgttttcaaagtact ctttcagttt ccattgtgaa gttttaagtg 60 atcgcgagtg ccaaaaagta acaatg gct gat gat cag gga cgt gga cgc agg 113 Met Ala Asp Asp Gln Gly ArgGly Arg Arg 1 5 10 cgt cca ntt aac gaa gat gat tcc tct act tcc cga ggtagt ggt gat 161 Arg Pro Xaa Asn Glu Asp Asp Ser Ser Thr Ser Arg Gly SerGly Asp 15 20 25 ggg ccg cgg gtg aaa gta ttc aga gga tct tca tca ggt gacccg aga 209 Gly Pro Arg Val Lys Val Phe Arg Gly Ser Ser Ser Gly Asp ProArg 30 35 40 gcg gat cct cgt ata gag gct tca aga gag aga aga gct ctc gaggaa 257 Ala Asp Pro Arg Ile Glu Ala Ser Arg Glu Arg Arg Ala Leu Glu Glu45 50 55 gct ccc agg cgt gaa ggt ggc ccg cca gag cga aag ccg tgg ggt gac305 Ala Pro Arg Arg Glu Gly Gly Pro Pro Glu Arg Lys Pro Trp Gly Asp 6065 70 caa tat gat tac ctg aat acc cgt ccg gtt gag ctg gta tcc aag aag353 Gln Tyr Asp Tyr Leu Asn Thr Arg Pro Val Glu Leu Val Ser Lys Lys 7580 85 90 gga acc gat ggc gtc ccg gtc atg ctg cag acg aac ttt ttt cga nta401 Gly Thr Asp Gly Val Pro Val Met Leu Gln Thr Asn Phe Phe Arg Xaa 95100 105 aaa acc aag ccg gaa tgg cgg atc gtt cat tat cac gtg gag ttt gtg449 Lys Thr Lys Pro Glu Trp Arg Ile Val His Tyr His Val Glu Phe Val 110115 120 ccg acc atc gag aat cct cgt gtc cgt atg gga gtt ttg tcc aat cat497 Pro Thr Ile Glu Asn Pro Arg Val Arg Met Gly Val Leu Ser Asn His 125130 135 gct aac ctt ctg gga tca ggc tat cta ttc gac gga ctg caa ctg ttc545 Ala Asn Leu Leu Gly Ser Gly Tyr Leu Phe Asp Gly Leu Gln Leu Phe 140145 150 acc acc agg aaa ttc gag cag gaa atc acg gtg ctc agc gga aag tcg593 Thr Thr Arg Lys Phe Glu Gln Glu Ile Thr Val Leu Ser Gly Lys Ser 155160 165 170 aag ctg gac att gaa tac aag ata tcc ata aag ttc gtt gga ttcata 641 Lys Leu Asp Ile Glu Tyr Lys Ile Ser Ile Lys Phe Val Gly Phe Ile175 180 185 tcg tgt gct gag ccc cgc ttt ttg caa gtc tta aat cta ata ttgcgc 689 Ser Cys Ala Glu Pro Arg Phe Leu Gln Val Leu Asn Leu Ile Leu Arg190 195 200 cgc tcg atg aag ggc cta aat ttg gaa tta gtt ggc cgt aat ctcttt 737 Arg Ser Met Lys Gly Leu Asn Leu Glu Leu Val Gly Arg Asn Leu Phe205 210 215 gat ccc cga gct aag atc gaa ata agg gag ttc aaa atg gag ctatgg 785 Asp Pro Arg Ala Lys Ile Glu Ile Arg Glu Phe Lys Met Glu Leu Trp220 225 230 ccg ggc tat gag aca tcg att cgt cag cac gaa aaa gat att ttattg 833 Pro Gly Tyr Glu Thr Ser Ile Arg Gln His Glu Lys Asp Ile Leu Leu235 240 245 250 ggc acc gaa ata act cac aaa gtt atg cgc acc gag acg atctac gac 881 Gly Thr Glu Ile Thr His Lys Val Met Arg Thr Glu Thr Ile TyrAsp 255 260 265 ata atg cga cgt tgc tca cac aat ccg gct cgt cat cag gacgaa gta 929 Ile Met Arg Arg Cys Ser His Asn Pro Ala Arg His Gln Asp GluVal 270 275 280 cgg gta aat gtt ttg gac ttg att gtc ctt acg gat tac aataac aga 977 Arg Val Asn Val Leu Asp Leu Ile Val Leu Thr Asp Tyr Asn AsnArg 285 290 295 act tat cgt atc aat gat gtc gac ttt gga caa act ccg aaatca aca 1025 Thr Tyr Arg Ile Asn Asp Val Asp Phe Gly Gln Thr Pro Lys SerThr 300 305 310 ttc agt tgc aag ggt aga gat atc agt ttc gtg gaa tac tatctc act 1073 Phe Ser Cys Lys Gly Arg Asp Ile Ser Phe Val Glu Tyr Tyr LeuThr 315 320 325 330 aaa tat aat ata cgc att cgc gac cac aat cag ccg ctgctg att tcc 1121 Lys Tyr Asn Ile Arg Ile Arg Asp His Asn Gln Pro Leu LeuIle Ser 335 340 345 aaa aat agg gac aag gct cta aaa act aac gct agc gaatta gtg gta 1169 Lys Asn Arg Asp Lys Ala Leu Lys Thr Asn Ala Ser Glu LeuVal Val 350 355 360 cta att cct gag ctc tgc cga gtg act ggg ctc aat gccgag atg cgc 1217 Leu Ile Pro Glu Leu Cys Arg Val Thr Gly Leu Asn Ala GluMet Arg 365 370 375 tca aac ttt cag ctt atg cgt gcc atg agc agt tat acgcga atg aac 1265 Ser Asn Phe Gln Leu Met Arg Ala Met Ser Ser Tyr Thr ArgMet Asn 380 385 390 ccc aaa caa cgc act gat cga ttg cgc gct ttt aac caccgt tta caa 1313 Pro Lys Gln Arg Thr Asp Arg Leu Arg Ala Phe Asn His ArgLeu Gln 395 400 405 410 aac act cca gaa agt gtg aag gtc ttg aga gac tggaac atg gaa ctg 1361 Asn Thr Pro Glu Ser Val Lys Val Leu Arg Asp Trp AsnMet Glu Leu 415 420 425 gac aag aac gtc aca gaa gta caa ggc cgg ata attgga cag cag aac 1409 Asp Lys Asn Val Thr Glu Val Gln Gly Arg Ile Ile GlyGln Gln Asn 430 435 440 atc gtg ttt cat aat gga aag gtt cct gct gga gaaaac gct gat tgg 1457 Ile Val Phe His Asn Gly Lys Val Pro Ala Gly Glu AsnAla Asp Trp 445 450 455 caa agg cac ttc aga gac caa agg atg ctt acc actccg agc gat ggc 1505 Gln Arg His Phe Arg Asp Gln Arg Met Leu Thr Thr ProSer Asp Gly 460 465 470 ctc gat cgt tgg gct gtc atc gcg ccg caa agg aattcc cat gaa ctc 1553 Leu Asp Arg Trp Ala Val Ile Ala Pro Gln Arg Asn SerHis Glu Leu 475 480 485 490 cga act cta ctt gac tct ttg tat aga gca gctagt gga atg ggt ctt 1601 Arg Thr Leu Leu Asp Ser Leu Tyr Arg Ala Ala SerGly Met Gly Leu 495 500 505 aga att cga agc ccc cag gaa ttc ata att tatgat gat cgc act gga 1649 Arg Ile Arg Ser Pro Gln Glu Phe Ile Ile Tyr AspAsp Arg Thr Gly 510 515 520 act tat gtg aga gca atg gat gat tgt gtg cgctca gat ccc aaa ctt 1697 Thr Tyr Val Arg Ala Met Asp Asp Cys Val Arg SerAsp Pro Lys Leu 525 530 535 ata tta tgc ctc gta ccc aat gat aac gcc gaaaga tac tca tca atc 1745 Ile Leu Cys Leu Val Pro Asn Asp Asn Ala Glu ArgTyr Ser Ser Ile 540 545 550 aaa aag aga gga tac gtt gac agg gcg gtg ccaact caa gtt gtg acc 1793 Lys Lys Arg Gly Tyr Val Asp Arg Ala Val Pro ThrGln Val Val Thr 555 560 565 570 ctt aaa acg acc aag aac cgt agc ctt atgagc att gcc acc aaa ata 1841 Leu Lys Thr Thr Lys Asn Arg Ser Leu Met SerIle Ala Thr Lys Ile 575 580 585 gca atc caa ctg aat tgc aag ttg gga tataca ccc tgg atg atc gaa 1889 Ala Ile Gln Leu Asn Cys Lys Leu Gly Tyr ThrPro Trp Met Ile Glu 590 595 600 cta ccc ttg tcc gga ctg atg aca att ggcttt gac att gcg aag agc 1937 Leu Pro Leu Ser Gly Leu Met Thr Ile Gly PheAsp Ile Ala Lys Ser 605 610 615 aca cga gat cgg aag agg gcc tac gga gcattg att gcc tca atg gat 1985 Thr Arg Asp Arg Lys Arg Ala Tyr Gly Ala LeuIle Ala Ser Met Asp 620 625 630 cta cag caa aac tcc acg tac ttc agc acagtc acg gag tgc agc gcc 2033 Leu Gln Gln Asn Ser Thr Tyr Phe Ser Thr ValThr Glu Cys Ser Ala 635 640 645 650 ttt gat gtg ctc gct aac acc ctt tggccg atg ata gca aag gcc ctg 2081 Phe Asp Val Leu Ala Asn Thr Leu Trp ProMet Ile Ala Lys Ala Leu 655 660 665 cgc caa tat caa cat gag cat agg aagctg cca tct cga atc gta ttt 2129 Arg Gln Tyr Gln His Glu His Arg Lys LeuPro Ser Arg Ile Val Phe 670 675 680 tat cga gac ggt gtg agc tcc ggc tctcta aag cag ctt ttt gaa ttt 2177 Tyr Arg Asp Gly Val Ser Ser Gly Ser LeuLys Gln Leu Phe Glu Phe 685 690 695 gaa gtc aag gac atc att gag aag ttgaaa act gaa tac gcc cgc gtc 2225 Glu Val Lys Asp Ile Ile Glu Lys Leu LysThr Glu Tyr Ala Arg Val 700 705 710 cag cta agc cca ccg caa tta gct tatatt gtg gta acc aga tcc atg 2273 Gln Leu Ser Pro Pro Gln Leu Ala Tyr IleVal Val Thr Arg Ser Met 715 720 725 730 aac acg cgc ttc ttc ctc aac ggacaa aat cct ccg cct ggt act ata 2321 Asn Thr Arg Phe Phe Leu Asn Gly GlnAsn Pro Pro Pro Gly Thr Ile 735 740 745 gtt gat gac gtt ata act ctg cccgag aga tac gac ttt tat ctg gtc 2369 Val Asp Asp Val Ile Thr Leu Pro GluArg Tyr Asp Phe Tyr Leu Val 750 755 760 tcg caa caa gtt cgt cag ggt acagtg tcg ccg acc agc tac aat gtt 2417 Ser Gln Gln Val Arg Gln Gly Thr ValSer Pro Thr Ser Tyr Asn Val 765 770 775 ctt tat agc agc atg ggt ntc tcaccg gag aaa atg caa aaa ctt acg 2465 Leu Tyr Ser Ser Met Gly Xaa Ser ProGlu Lys Met Gln Lys Leu Thr 780 785 790 tac aag atg tgc cac ttg tac tacaat tgg tcg ggc acc aca cga gtg 2513 Tyr Lys Met Cys His Leu Tyr Tyr AsnTrp Ser Gly Thr Thr Arg Val 795 800 805 810 cca gca gtt tgc cag tac gctaag aag cta gct acc ctc gtg ggt acg 2561 Pro Ala Val Cys Gln Tyr Ala LysLys Leu Ala Thr Leu Val Gly Thr 815 820 825 aac ttg cac tct att ccg caaaac gcg ctc gaa aag aag ttt tat tat 2609 Asn Leu His Ser Ile Pro Gln AsnAla Leu Glu Lys Lys Phe Tyr Tyr 830 835 840 cta taattggata taatttagaatggagtatta atccttacta agaggccata 2662 Leu tatgaaacta gcccagacatttatactttt tcaatacttc cttacttttg ctaagcactt 2722 cagcatttat gactaaatattttgtatttg aaatgcatta ctgctctttt ttcaaacaaa 2782 agcaaaattg aggattaagattctggtatt taagcataag accagaggaa attcccaaac 2842 aaacatttaa agttatctatcaagacatgt tcattaattt ggaatataat tactttattt 2902 tttattgtat attttagtttatgtaaagaa aaattacata catccatgtt tgcttactta 2962 accacacatt catggctgcttatattcgtg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3022 aaaaaaaaaa aaaaaaaaaaaaaaa 3047 2 843 PRT Drosophila sp. misc_feature (13)..(13) The ′Xaa′ atlocation 13 stands for Ile, Val, Leu, or Phe. 2 Met Ala Asp Asp Gln GlyArg Gly Arg Arg Arg Pro Xaa Asn Glu Asp 1 5 10 15 Asp Ser Ser Thr SerArg Gly Ser Gly Asp Gly Pro Arg Val Lys Val 20 25 30 Phe Arg Gly Ser SerSer Gly Asp Pro Arg Ala Asp Pro Arg Ile Glu 35 40 45 Ala Ser Arg Glu ArgArg Ala Leu Glu Glu Ala Pro Arg Arg Glu Gly 50 55 60 Gly Pro Pro Glu ArgLys Pro Trp Gly Asp Gln Tyr Asp Tyr Leu Asn 65 70 75 80 Thr Arg Pro ValGlu Leu Val Ser Lys Lys Gly Thr Asp Gly Val Pro 85 90 95 Val Met Leu GlnThr Asn Phe Phe Arg Xaa Lys Thr Lys Pro Glu Trp 100 105 110 Arg Ile ValHis Tyr His Val Glu Phe Val Pro Thr Ile Glu Asn Pro 115 120 125 Arg ValArg Met Gly Val Leu Ser Asn His Ala Asn Leu Leu Gly Ser 130 135 140 GlyTyr Leu Phe Asp Gly Leu Gln Leu Phe Thr Thr Arg Lys Phe Glu 145 150 155160 Gln Glu Ile Thr Val Leu Ser Gly Lys Ser Lys Leu Asp Ile Glu Tyr 165170 175 Lys Ile Ser Ile Lys Phe Val Gly Phe Ile Ser Cys Ala Glu Pro Arg180 185 190 Phe Leu Gln Val Leu Asn Leu Ile Leu Arg Arg Ser Met Lys GlyLeu 195 200 205 Asn Leu Glu Leu Val Gly Arg Asn Leu Phe Asp Pro Arg AlaLys Ile 210 215 220 Glu Ile Arg Glu Phe Lys Met Glu Leu Trp Pro Gly TyrGlu Thr Ser 225 230 235 240 Ile Arg Gln His Glu Lys Asp Ile Leu Leu GlyThr Glu Ile Thr His 245 250 255 Lys Val Met Arg Thr Glu Thr Ile Tyr AspIle Met Arg Arg Cys Ser 260 265 270 His Asn Pro Ala Arg His Gln Asp GluVal Arg Val Asn Val Leu Asp 275 280 285 Leu Ile Val Leu Thr Asp Tyr AsnAsn Arg Thr Tyr Arg Ile Asn Asp 290 295 300 Val Asp Phe Gly Gln Thr ProLys Ser Thr Phe Ser Cys Lys Gly Arg 305 310 315 320 Asp Ile Ser Phe ValGlu Tyr Tyr Leu Thr Lys Tyr Asn Ile Arg Ile 325 330 335 Arg Asp His AsnGln Pro Leu Leu Ile Ser Lys Asn Arg Asp Lys Ala 340 345 350 Leu Lys ThrAsn Ala Ser Glu Leu Val Val Leu Ile Pro Glu Leu Cys 355 360 365 Arg ValThr Gly Leu Asn Ala Glu Met Arg Ser Asn Phe Gln Leu Met 370 375 380 ArgAla Met Ser Ser Tyr Thr Arg Met Asn Pro Lys Gln Arg Thr Asp 385 390 395400 Arg Leu Arg Ala Phe Asn His Arg Leu Gln Asn Thr Pro Glu Ser Val 405410 415 Lys Val Leu Arg Asp Trp Asn Met Glu Leu Asp Lys Asn Val Thr Glu420 425 430 Val Gln Gly Arg Ile Ile Gly Gln Gln Asn Ile Val Phe His AsnGly 435 440 445 Lys Val Pro Ala Gly Glu Asn Ala Asp Trp Gln Arg His PheArg Asp 450 455 460 Gln Arg Met Leu Thr Thr Pro Ser Asp Gly Leu Asp ArgTrp Ala Val 465 470 475 480 Ile Ala Pro Gln Arg Asn Ser His Glu Leu ArgThr Leu Leu Asp Ser 485 490 495 Leu Tyr Arg Ala Ala Ser Gly Met Gly LeuArg Ile Arg Ser Pro Gln 500 505 510 Glu Phe Ile Ile Tyr Asp Asp Arg ThrGly Thr Tyr Val Arg Ala Met 515 520 525 Asp Asp Cys Val Arg Ser Asp ProLys Leu Ile Leu Cys Leu Val Pro 530 535 540 Asn Asp Asn Ala Glu Arg TyrSer Ser Ile Lys Lys Arg Gly Tyr Val 545 550 555 560 Asp Arg Ala Val ProThr Gln Val Val Thr Leu Lys Thr Thr Lys Asn 565 570 575 Arg Ser Leu MetSer Ile Ala Thr Lys Ile Ala Ile Gln Leu Asn Cys 580 585 590 Lys Leu GlyTyr Thr Pro Trp Met Ile Glu Leu Pro Leu Ser Gly Leu 595 600 605 Met ThrIle Gly Phe Asp Ile Ala Lys Ser Thr Arg Asp Arg Lys Arg 610 615 620 AlaTyr Gly Ala Leu Ile Ala Ser Met Asp Leu Gln Gln Asn Ser Thr 625 630 635640 Tyr Phe Ser Thr Val Thr Glu Cys Ser Ala Phe Asp Val Leu Ala Asn 645650 655 Thr Leu Trp Pro Met Ile Ala Lys Ala Leu Arg Gln Tyr Gln His Glu660 665 670 His Arg Lys Leu Pro Ser Arg Ile Val Phe Tyr Arg Asp Gly ValSer 675 680 685 Ser Gly Ser Leu Lys Gln Leu Phe Glu Phe Glu Val Lys AspIle Ile 690 695 700 Glu Lys Leu Lys Thr Glu Tyr Ala Arg Val Gln Leu SerPro Pro Gln 705 710 715 720 Leu Ala Tyr Ile Val Val Thr Arg Ser Met AsnThr Arg Phe Phe Leu 725 730 735 Asn Gly Gln Asn Pro Pro Pro Gly Thr IleVal Asp Asp Val Ile Thr 740 745 750 Leu Pro Glu Arg Tyr Asp Phe Tyr LeuVal Ser Gln Gln Val Arg Gln 755 760 765 Gly Thr Val Ser Pro Thr Ser TyrAsn Val Leu Tyr Ser Ser Met Gly 770 775 780 Xaa Ser Pro Glu Lys Met GlnLys Leu Thr Tyr Lys Met Cys His Leu 785 790 795 800 Tyr Tyr Asn Trp SerGly Thr Thr Arg Val Pro Ala Val Cys Gln Tyr 805 810 815 Ala Lys Lys LeuAla Thr Leu Val Gly Thr Asn Leu His Ser Ile Pro 820 825 830 Gln Asn AlaLeu Glu Lys Lys Phe Tyr Tyr Leu 835 840 3 4064 DNA Mus sp. CDS(191)..(2776) misc_feature (458)..(458) n = a or c; Xaa = Leu or Ile 3agaagcctgc tacagggggt ggcgtgagga gcctcaagga ctgcggacct aagcgtgcta 60agcacggggc caggatagga ctggacgctg ctggaccacg acgatcaggg agtgaccccc 120accagcatcc agcctgcggc agttgtagga ctggaaggag gccaagctga acacctgagc 180tgactggaaa atg act ggc cga gcc cga gct cgg gcc cgc ggc agg gca 229 MetThr Gly Arg Ala Arg Ala Arg Ala Arg Gly Arg Ala 1 5 10 cga ggt cag gagacg gtg cag cat gtt ggg gct gct gcg agc cag caa 277 Arg Gly Gln Glu ThrVal Gln His Val Gly Ala Ala Ala Ser Gln Gln 15 20 25 cct ggg tac atc ccaccg aga cct caa cag tcc ccc aca gag ggg gac 325 Pro Gly Tyr Ile Pro ProArg Pro Gln Gln Ser Pro Thr Glu Gly Asp 30 35 40 45 ttg gtt ggc cga ggacga cag agg ggg atg gta gtc gga gcc aca tcc 373 Leu Val Gly Arg Gly ArgGln Arg Gly Met Val Val Gly Ala Thr Ser 50 55 60 aag tca caa gaa ctg cagatc tca gct ggg ttt cag gag ctg tca ctg 421 Lys Ser Gln Glu Leu Gln IleSer Ala Gly Phe Gln Glu Leu Ser Leu 65 70 75 gca gag aga gga ggg cgt cgccga gac ttc cat gac ntt ggt gtg aac 469 Ala Glu Arg Gly Gly Arg Arg ArgAsp Phe His Asp Xaa Gly Val Asn 80 85 90 acc aga cag aac ctt gac cat gtcaaa gag tca aag aca ggc tcc tct 517 Thr Arg Gln Asn Leu Asp His Val LysGlu Ser Lys Thr Gly Ser Ser 95 100 105 ggc atc att gtg aag ctg agc accaac cac ttc cgg ctg acc tcg cgc 565 Gly Ile Ile Val Lys Leu Ser Thr AsnHis Phe Arg Leu Thr Ser Arg 110 115 120 125 cca cag tgg gcc ctg tat cagtac cac atc gac tac aat ccc ctg atg 613 Pro Gln Trp Ala Leu Tyr Gln TyrHis Ile Asp Tyr Asn Pro Leu Met 130 135 140 gag gcc cga agg ctt cgc tccgca ctg ctc ttc cag cat gaa gac ctc 661 Glu Ala Arg Arg Leu Arg Ser AlaLeu Leu Phe Gln His Glu Asp Leu 145 150 155 att gga agg tgt cat gct ttcgat ggg aca ata ttg ttt tta cct aag 709 Ile Gly Arg Cys His Ala Phe AspGly Thr Ile Leu Phe Leu Pro Lys 160 165 170 aga cta cag cac aag gtc acagaa gta ttc agt cag act cgg aat ggg 757 Arg Leu Gln His Lys Val Thr GluVal Phe Ser Gln Thr Arg Asn Gly 175 180 185 gaa cac gtg agg atc acc atcacc ctg acc aac gag ctg ccg ccc acc 805 Glu His Val Arg Ile Thr Ile ThrLeu Thr Asn Glu Leu Pro Pro Thr 190 195 200 205 tcg ccc acc tgc ctg cagttc tat aac atc ntc ttc agg agg ctc ttg 853 Ser Pro Thr Cys Leu Gln PheTyr Asn Ile Xaa Phe Arg Arg Leu Leu 210 215 220 aaa atc atg aat ttg caacaa att gga cgg aat tat tac aat cca agt 901 Lys Ile Met Asn Leu Gln GlnIle Gly Arg Asn Tyr Tyr Asn Pro Ser 225 230 235 gac ccg att gat att ccaaac cac agg ttg gtg atc tgg ccc ggc ttc 949 Asp Pro Ile Asp Ile Pro AsnHis Arg Leu Val Ile Trp Pro Gly Phe 240 245 250 acc acc tcc atc ctt cagtat gag aac aac atc atg ctc tgc aca gac 997 Thr Thr Ser Ile Leu Gln TyrGlu Asn Asn Ile Met Leu Cys Thr Asp 255 260 265 gtc agc cac aag gtg ctccgc agc gag act gtc cta gac ttc atg ttc 1045 Val Ser His Lys Val Leu ArgSer Glu Thr Val Leu Asp Phe Met Phe 270 275 280 285 aat cta tac cag cagaca gag gag cac aag ttc cag gag caa gtg tcg 1093 Asn Leu Tyr Gln Gln ThrGlu Glu His Lys Phe Gln Glu Gln Val Ser 290 295 300 aag gag ctc ata ggcctc atc gtt ctc acc aag tac aat aac aag acc 1141 Lys Glu Leu Ile Gly LeuIle Val Leu Thr Lys Tyr Asn Asn Lys Thr 305 310 315 tac cgg gtg gat gacatt gac tgg gac cag aat cca aag agc acc ttc 1189 Tyr Arg Val Asp Asp IleAsp Trp Asp Gln Asn Pro Lys Ser Thr Phe 320 325 330 aag aag gcg gat ggctcg gag gtc agc ttc ctg gag tac tac agg aag 1237 Lys Lys Ala Asp Gly SerGlu Val Ser Phe Leu Glu Tyr Tyr Arg Lys 335 340 345 caa tac aac cag gagatc acg gac ctg aag cag ccg gtg ctg gtg agc 1285 Gln Tyr Asn Gln Glu IleThr Asp Leu Lys Gln Pro Val Leu Val Ser 350 355 360 365 caa ccc aag cggagg aga ggc ccc ggc ggc acc ctg cct ggc cca gct 1333 Gln Pro Lys Arg ArgArg Gly Pro Gly Gly Thr Leu Pro Gly Pro Ala 370 375 380 atg ntc atc cctgaa ctc tgc tat ctc aca ggc ctg act gat aaa atg 1381 Met Xaa Ile Pro GluLeu Cys Tyr Leu Thr Gly Leu Thr Asp Lys Met 385 390 395 cgc aat gat ttcaat gtg atg aag gac ctg gca gtg cac acg cgg ctg 1429 Arg Asn Asp Phe AsnVal Met Lys Asp Leu Ala Val His Thr Arg Leu 400 405 410 acc cct gag cagcgg cag cgg gag gtg ggc cgc ctc atc gac tac atc 1477 Thr Pro Glu Gln ArgGln Arg Glu Val Gly Arg Leu Ile Asp Tyr Ile 415 420 425 cac aag gat gacaat gtg cag aga gag ctt cga gac tgg ggc ctg agc 1525 His Lys Asp Asp AsnVal Gln Arg Glu Leu Arg Asp Trp Gly Leu Ser 430 435 440 445 ttc gac tcaaac ttg ctg tcc ttc tct gga aga atc tta caa tct gag 1573 Phe Asp Ser AsnLeu Leu Ser Phe Ser Gly Arg Ile Leu Gln Ser Glu 450 455 460 aag atc caccag ggc gga aag acg ttt gat tac aac cca caa ttt gca 1621 Lys Ile His GlnGly Gly Lys Thr Phe Asp Tyr Asn Pro Gln Phe Ala 465 470 475 gac tgg tccaaa gaa aca aga ggc gcg ccg ctg atc agc gtg aag cca 1669 Asp Trp Ser LysGlu Thr Arg Gly Ala Pro Leu Ile Ser Val Lys Pro 480 485 490 ttg gat aactgg ctg ctg atc tat acc cgc agg aat tat gaa gca gcc 1717 Leu Asp Asn TrpLeu Leu Ile Tyr Thr Arg Arg Asn Tyr Glu Ala Ala 495 500 505 aac tca ctgata cag aac ctg ttc aaa gtg act cca gcc atg ggc atc 1765 Asn Ser Leu IleGln Asn Leu Phe Lys Val Thr Pro Ala Met Gly Ile 510 515 520 525 cag atgaaa aag gca atc atg atc gag gtg gat gac aga aca gaa gct 1813 Gln Met LysLys Ala Ile Met Ile Glu Val Asp Asp Arg Thr Glu Ala 530 535 540 tat ctgaga gcc ttg cag cag aag gtg acg tca gac act cag ata gtt 1861 Tyr Leu ArgAla Leu Gln Gln Lys Val Thr Ser Asp Thr Gln Ile Val 545 550 555 gtc tgtctc ttg tca agt aat cgg aag gac aaa tat gat gcc atc aag 1909 Val Cys LeuLeu Ser Ser Asn Arg Lys Asp Lys Tyr Asp Ala Ile Lys 560 565 570 aag tacttg tgt aca gac tgc ccc acc cca agt cag tgt gtg gtg gcc 1957 Lys Tyr LeuCys Thr Asp Cys Pro Thr Pro Ser Gln Cys Val Val Ala 575 580 585 cgg accctg ggc aag cag caa aca gtc atg gcc att gcc acc aag atc 2005 Arg Thr LeuGly Lys Gln Gln Thr Val Met Ala Ile Ala Thr Lys Ile 590 595 600 605 gccctg cag atg aac tgc aag atg gga ggc gag ctc tgg cgg gtg gac 2053 Ala LeuGln Met Asn Cys Lys Met Gly Gly Glu Leu Trp Arg Val Asp 610 615 620 atggcc ctg aaa ctg gca atg atc gtg ggc atc gac tgt tac cat gac 2101 Met AlaLeu Lys Leu Ala Met Ile Val Gly Ile Asp Cys Tyr His Asp 625 630 635 accaca gct ggg cgg agg tcc atc gca gga ttc gtc gcc agc atc aat 2149 Thr ThrAla Gly Arg Arg Ser Ile Ala Gly Phe Val Ala Ser Ile Asn 640 645 650 gaaggg atg acc cgc tgg ttc tcc cgc tgc gtc ttt cag gac cgc ggg 2197 Glu GlyMet Thr Arg Trp Phe Ser Arg Cys Val Phe Gln Asp Arg Gly 655 660 665 caggag ctg gtg gat ggt ctc aag gtg tgc ttg caa gct gct ctg agg 2245 Gln GluLeu Val Asp Gly Leu Lys Val Cys Leu Gln Ala Ala Leu Arg 670 675 680 685gct tgg agt ggc tgc aat gaa tac atg ccc agc cgt gtc atc gtg tac 2293 AlaTrp Ser Gly Cys Asn Glu Tyr Met Pro Ser Arg Val Ile Val Tyr 690 695 700cga gac ggt gtg ggg gac ggg cag ctg aag acc ctg gtc aat tat gag 2341 ArgAsp Gly Val Gly Asp Gly Gln Leu Lys Thr Leu Val Asn Tyr Glu 705 710 715gtc cca cag ttc cta gat tgc ctc aag tca gtc ggg aga ggt tac aac 2389 ValPro Gln Phe Leu Asp Cys Leu Lys Ser Val Gly Arg Gly Tyr Asn 720 725 730cca aga ctg act gta atc gtg gtg aag aag cgt gtc aat gcc agg ttt 2437 ProArg Leu Thr Val Ile Val Val Lys Lys Arg Val Asn Ala Arg Phe 735 740 745ttt gct cag tct ggg gga aga ctt cag aac cct ctt cca ggg aca gtc 2485 PheAla Gln Ser Gly Gly Arg Leu Gln Asn Pro Leu Pro Gly Thr Val 750 755 760765 atc gat gtg gaa gtc acc aga cca gag tgg tat gac ttt ttc atc gtg 2533Ile Asp Val Glu Val Thr Arg Pro Glu Trp Tyr Asp Phe Phe Ile Val 770 775780 agc cag gca gtg aga agc ggg agt gtg tcc cca aca cac tac aat gtc 2581Ser Gln Ala Val Arg Ser Gly Ser Val Ser Pro Thr His Tyr Asn Val 785 790795 atc tat gac agc agt ggc ctg aag ccc gac cac atc cag cgg ctg aca 2629Ile Tyr Asp Ser Ser Gly Leu Lys Pro Asp His Ile Gln Arg Leu Thr 800 805810 tac aag ntc tgc cac gtg tac tat aat tgg cct gga gtc atc cga gtc 2677Tyr Lys Xaa Cys His Val Tyr Tyr Asn Trp Pro Gly Val Ile Arg Val 815 820825 cct gca cct tgc cag tat gca cac aag ctg gcc ttc ctc gtg ggc cag 2725Pro Ala Pro Cys Gln Tyr Ala His Lys Leu Ala Phe Leu Val Gly Gln 830 835840 845 agc atc cac aga gag cca aac ctc tcc ctg tcc aac cgc ctc tac tac2773 Ser Ile His Arg Glu Pro Asn Leu Ser Leu Ser Asn Arg Leu Tyr Tyr 850855 860 ctc taaccagcag cgagggccgg catgctggtt tccttcagag ctctgcccaa 2826Leu tccttcagtt ctatttttca taggatgagg ttctagcggg atgttggggg taggggttgg2886 gggccgctgt tttgttttgt tttgttttta gttgttggtg tgtagcggct atagagttaa2946 ctaaggctta tgtttgcttt gtttctcatg gtgtgttgtg acagtttgct tggcagagtg3006 gtggcaaggg ccgtggtacc acagatagac tttcctaaag cagccgtgtt ggcaggttct3066 tggaggtgct cctgggagca cctcctattg tacgctgctc tctctctctc tctctctctc3126 tctctctctc tctctctctc tctctctctc tgcatgtttg aatttaaaag aggaaaggac3186 acaaggcaag aatgcccaga gcatcctaga tgggtgctat atgggactct gacacctcag3246 gttcttaaag ataaaaaggc cagaggtacc tttctgtaat tcttttaaga gttaggtcat3306 aaggaatgaa aagagttact tttctccctc catgaaagtg ttttttaaga ctgacaaggc3366 cggtggtgga gtgagtgttg ttgttttgtt tcgcaatttt cttaccaagt gaaattactg3426 accataattt tatttaatag tttttagttt ttgaaagcct gtgttttctt tgcttttaaa3486 ctacaggcga ccactctggg ataggattcc actcccaaaa gtaatgtgtt gggtgtggtg3546 gcacacgcct gtgatctcag gactcaggag gtgaagccag gggaacccca agttcccggt3606 cagcctaggc tacatagtga gactttgtct taacaaacag aaaaaaagaa aaagaaagaa3666 aaaaaaaaga aagaaaaaaa aaraaagaaa aatggagcag tatgagttat gggtgccagt3726 ttctgcagga gactttggga gtaagagctg cctctgttcc taagaggctg caaggctgcg3786 gctggggggc cagttcaggt gccaggtgag gtacggccac atctatttgc ctggctcttt3846 gacaaagaga gcagcttaga gtggctagag tgtgatgagt tttacacaat ggcgatgtgt3906 gtgtgtgccc ctggcacagc tcaataaagt ttctgtctca ctttaaaaaa aaaaaaaaaa3966 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa4026 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 4064 4 862 PRT Mus sp.misc_feature (90)..(90) The ′Xaa′ at location 90 stands for Ile, Val,Leu, or Phe. 4 Met Thr Gly Arg Ala Arg Ala Arg Ala Arg Gly Arg Ala ArgGly Gln 1 5 10 15 Glu Thr Val Gln His Val Gly Ala Ala Ala Ser Gln GlnPro Gly Tyr 20 25 30 Ile Pro Pro Arg Pro Gln Gln Ser Pro Thr Glu Gly AspLeu Val Gly 35 40 45 Arg Gly Arg Gln Arg Gly Met Val Val Gly Ala Thr SerLys Ser Gln 50 55 60 Glu Leu Gln Ile Ser Ala Gly Phe Gln Glu Leu Ser LeuAla Glu Arg 65 70 75 80 Gly Gly Arg Arg Arg Asp Phe His Asp Xaa Gly ValAsn Thr Arg Gln 85 90 95 Asn Leu Asp His Val Lys Glu Ser Lys Thr Gly SerSer Gly Ile Ile 100 105 110 Val Lys Leu Ser Thr Asn His Phe Arg Leu ThrSer Arg Pro Gln Trp 115 120 125 Ala Leu Tyr Gln Tyr His Ile Asp Tyr AsnPro Leu Met Glu Ala Arg 130 135 140 Arg Leu Arg Ser Ala Leu Leu Phe GlnHis Glu Asp Leu Ile Gly Arg 145 150 155 160 Cys His Ala Phe Asp Gly ThrIle Leu Phe Leu Pro Lys Arg Leu Gln 165 170 175 His Lys Val Thr Glu ValPhe Ser Gln Thr Arg Asn Gly Glu His Val 180 185 190 Arg Ile Thr Ile ThrLeu Thr Asn Glu Leu Pro Pro Thr Ser Pro Thr 195 200 205 Cys Leu Gln PheTyr Asn Ile Xaa Phe Arg Arg Leu Leu Lys Ile Met 210 215 220 Asn Leu GlnGln Ile Gly Arg Asn Tyr Tyr Asn Pro Ser Asp Pro Ile 225 230 235 240 AspIle Pro Asn His Arg Leu Val Ile Trp Pro Gly Phe Thr Thr Ser 245 250 255Ile Leu Gln Tyr Glu Asn Asn Ile Met Leu Cys Thr Asp Val Ser His 260 265270 Lys Val Leu Arg Ser Glu Thr Val Leu Asp Phe Met Phe Asn Leu Tyr 275280 285 Gln Gln Thr Glu Glu His Lys Phe Gln Glu Gln Val Ser Lys Glu Leu290 295 300 Ile Gly Leu Ile Val Leu Thr Lys Tyr Asn Asn Lys Thr Tyr ArgVal 305 310 315 320 Asp Asp Ile Asp Trp Asp Gln Asn Pro Lys Ser Thr PheLys Lys Ala 325 330 335 Asp Gly Ser Glu Val Ser Phe Leu Glu Tyr Tyr ArgLys Gln Tyr Asn 340 345 350 Gln Glu Ile Thr Asp Leu Lys Gln Pro Val LeuVal Ser Gln Pro Lys 355 360 365 Arg Arg Arg Gly Pro Gly Gly Thr Leu ProGly Pro Ala Met Xaa Ile 370 375 380 Pro Glu Leu Cys Tyr Leu Thr Gly LeuThr Asp Lys Met Arg Asn Asp 385 390 395 400 Phe Asn Val Met Lys Asp LeuAla Val His Thr Arg Leu Thr Pro Glu 405 410 415 Gln Arg Gln Arg Glu ValGly Arg Leu Ile Asp Tyr Ile His Lys Asp 420 425 430 Asp Asn Val Gln ArgGlu Leu Arg Asp Trp Gly Leu Ser Phe Asp Ser 435 440 445 Asn Leu Leu SerPhe Ser Gly Arg Ile Leu Gln Ser Glu Lys Ile His 450 455 460 Gln Gly GlyLys Thr Phe Asp Tyr Asn Pro Gln Phe Ala Asp Trp Ser 465 470 475 480 LysGlu Thr Arg Gly Ala Pro Leu Ile Ser Val Lys Pro Leu Asp Asn 485 490 495Trp Leu Leu Ile Tyr Thr Arg Arg Asn Tyr Glu Ala Ala Asn Ser Leu 500 505510 Ile Gln Asn Leu Phe Lys Val Thr Pro Ala Met Gly Ile Gln Met Lys 515520 525 Lys Ala Ile Met Ile Glu Val Asp Asp Arg Thr Glu Ala Tyr Leu Arg530 535 540 Ala Leu Gln Gln Lys Val Thr Ser Asp Thr Gln Ile Val Val CysLeu 545 550 555 560 Leu Ser Ser Asn Arg Lys Asp Lys Tyr Asp Ala Ile LysLys Tyr Leu 565 570 575 Cys Thr Asp Cys Pro Thr Pro Ser Gln Cys Val ValAla Arg Thr Leu 580 585 590 Gly Lys Gln Gln Thr Val Met Ala Ile Ala ThrLys Ile Ala Leu Gln 595 600 605 Met Asn Cys Lys Met Gly Gly Glu Leu TrpArg Val Asp Met Ala Leu 610 615 620 Lys Leu Ala Met Ile Val Gly Ile AspCys Tyr His Asp Thr Thr Ala 625 630 635 640 Gly Arg Arg Ser Ile Ala GlyPhe Val Ala Ser Ile Asn Glu Gly Met 645 650 655 Thr Arg Trp Phe Ser ArgCys Val Phe Gln Asp Arg Gly Gln Glu Leu 660 665 670 Val Asp Gly Leu LysVal Cys Leu Gln Ala Ala Leu Arg Ala Trp Ser 675 680 685 Gly Cys Asn GluTyr Met Pro Ser Arg Val Ile Val Tyr Arg Asp Gly 690 695 700 Val Gly AspGly Gln Leu Lys Thr Leu Val Asn Tyr Glu Val Pro Gln 705 710 715 720 PheLeu Asp Cys Leu Lys Ser Val Gly Arg Gly Tyr Asn Pro Arg Leu 725 730 735Thr Val Ile Val Val Lys Lys Arg Val Asn Ala Arg Phe Phe Ala Gln 740 745750 Ser Gly Gly Arg Leu Gln Asn Pro Leu Pro Gly Thr Val Ile Asp Val 755760 765 Glu Val Thr Arg Pro Glu Trp Tyr Asp Phe Phe Ile Val Ser Gln Ala770 775 780 Val Arg Ser Gly Ser Val Ser Pro Thr His Tyr Asn Val Ile TyrAsp 785 790 795 800 Ser Ser Gly Leu Lys Pro Asp His Ile Gln Arg Leu ThrTyr Lys Xaa 805 810 815 Cys His Val Tyr Tyr Asn Trp Pro Gly Val Ile ArgVal Pro Ala Pro 820 825 830 Cys Gln Tyr Ala His Lys Leu Ala Phe Leu ValGly Gln Ser Ile His 835 840 845 Arg Glu Pro Asn Leu Ser Leu Ser Asn ArgLeu Tyr Tyr Leu 850 855 860 5 3472 DNA Homo sapiens CDS (167)..(2749)misc_feature (392)..(392) n = t or a; Xaa = Leu or Ile 5 gggcgaattgggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat 60 tccatcctaatacgactcac tatagggctc gagcggccgc ccgggcaggt ctgaggtgca 120 aggaccaggactagggcgag ggcagcggtc caagaaatag aaaaca atg act ggg 175 Met Thr Gly 1aga gcc cga gcc aga gcc aga gga agg gcc cgc ggt cag gag aca gcg 223 ArgAla Arg Ala Arg Ala Arg Gly Arg Ala Arg Gly Gln Glu Thr Ala 5 10 15 cagctg gtg ggc tcc act gcc agt cag caa cct ggt tat att cag cct 271 Gln LeuVal Gly Ser Thr Ala Ser Gln Gln Pro Gly Tyr Ile Gln Pro 20 25 30 35 aggcct cag ccg cca cca gca gag ggg gaa tta ttt ggc cgt gga cgg 319 Arg ProGln Pro Pro Pro Ala Glu Gly Glu Leu Phe Gly Arg Gly Arg 40 45 50 cag agagga aca gca gga gga aca gcc aag tca caa gga ctc cag ata 367 Gln Arg GlyThr Ala Gly Gly Thr Ala Lys Ser Gln Gly Leu Gln Ile 55 60 65 tct gct ggattt cag gag tta tcg nta gca gag aga gga ggt cgt cgt 415 Ser Ala Gly PheGln Glu Leu Ser Xaa Ala Glu Arg Gly Gly Arg Arg 70 75 80 aga gat ttt catgat ctt ggt gtg aat aca agg cag aac cta gac cat 463 Arg Asp Phe His AspLeu Gly Val Asn Thr Arg Gln Asn Leu Asp His 85 90 95 gtt aaa gaa tca aaaaca ggt tct tca ggc att ata gta agg tta agc 511 Val Lys Glu Ser Lys ThrGly Ser Ser Gly Ile Ile Val Arg Leu Ser 100 105 110 115 act aac cat ttccgg ctg aca tcc cgt ccc cag tgg gcc tta tat cag 559 Thr Asn His Phe ArgLeu Thr Ser Arg Pro Gln Trp Ala Leu Tyr Gln 120 125 130 tat cac att gactat aac cca ctg atg gaa gcc aga aga ctc cgt tca 607 Tyr His Ile Asp TyrAsn Pro Leu Met Glu Ala Arg Arg Leu Arg Ser 135 140 145 gct ctt ctt tttcaa cac gaa gat cta att gga aag tgt cat gct ttt 655 Ala Leu Leu Phe GlnHis Glu Asp Leu Ile Gly Lys Cys His Ala Phe 150 155 160 gat gga acg atatta ttt tta cct aaa aga cta cag caa aag gtt act 703 Asp Gly Thr Ile LeuPhe Leu Pro Lys Arg Leu Gln Gln Lys Val Thr 165 170 175 gaa gtt ttt agtaag acc cgg aat gga gag gat gtg agg ata acg atc 751 Glu Val Phe Ser LysThr Arg Asn Gly Glu Asp Val Arg Ile Thr Ile 180 185 190 195 act tta acaaat gaa ctt cca cct aca tca cca act tgt ttg cag ttc 799 Thr Leu Thr AsnGlu Leu Pro Pro Thr Ser Pro Thr Cys Leu Gln Phe 200 205 210 tat aat attatt ttc agg agg ctt ttg aaa atc atg aat ttg caa caa 847 Tyr Asn Ile IlePhe Arg Arg Leu Leu Lys Ile Met Asn Leu Gln Gln 215 220 225 att gga cgaaat tat tat aac cca aat gac cca att gat att cca agt 895 Ile Gly Arg AsnTyr Tyr Asn Pro Asn Asp Pro Ile Asp Ile Pro Ser 230 235 240 cac agg ttggtg att tgg cct ggc ttc act act tcc atc ctt cag tat 943 His Arg Leu ValIle Trp Pro Gly Phe Thr Thr Ser Ile Leu Gln Tyr 245 250 255 gaa aac agcatc atg ctc tgc act gac gtt agc cat aaa gtc ctt cga 991 Glu Asn Ser IleMet Leu Cys Thr Asp Val Ser His Lys Val Leu Arg 260 265 270 275 agt gagact gtt ttg gat ttc atg ttc aac ttt tat cat cag aca gaa 1039 Ser Glu ThrVal Leu Asp Phe Met Phe Asn Phe Tyr His Gln Thr Glu 280 285 290 gaa cataaa ttt caa gaa caa gtt tcc aaa gaa nta ata ggt tta gtt 1087 Glu His LysPhe Gln Glu Gln Val Ser Lys Glu Xaa Ile Gly Leu Val 295 300 305 gtt cttacc aag tat aac att aag aca tac aga gtg gat gat att gac 1135 Val Leu ThrLys Tyr Asn Ile Lys Thr Tyr Arg Val Asp Asp Ile Asp 310 315 320 tgg gaccag aat ccc aag agc acc ttt aag aaa gcc gac ggc tct ggg 1183 Trp Asp GlnAsn Pro Lys Ser Thr Phe Lys Lys Ala Asp Gly Ser Gly 325 330 335 gtc agcttc tta gaa tac tac agg aag caa tac aac caa gag atc acc 1231 Val Ser PheLeu Glu Tyr Tyr Arg Lys Gln Tyr Asn Gln Glu Ile Thr 340 345 350 355 gacttg aag cag cct gtc ttg gtc agc cag ccc aag aga agg cgg ggc 1279 Asp LeuLys Gln Pro Val Leu Val Ser Gln Pro Lys Arg Arg Arg Gly 360 365 370 cctggg ggg aca ctg cca ggg cct gcc atg ctc att cct gag ctc tgc 1327 Pro GlyGly Thr Leu Pro Gly Pro Ala Met Leu Ile Pro Glu Leu Cys 375 380 385 tatctt aca ggt cta act gat aaa atg cgt aat gat ttt aac gtg atg 1375 Tyr LeuThr Gly Leu Thr Asp Lys Met Arg Asn Asp Phe Asn Val Met 390 395 400 aaagac tta gcc gtt cat aca aga cta act cca gag caa agg cag cgt 1423 Lys AspLeu Ala Val His Thr Arg Leu Thr Pro Glu Gln Arg Gln Arg 405 410 415 gaagtg gga cga ctc att gat tac att cat aaa aac gat aat gtt caa 1471 Glu ValGly Arg Leu Ile Asp Tyr Ile His Lys Asn Asp Asn Val Gln 420 425 430 435agg gag ctt cga gac tgg ggt ttg agc ttt gat tcc aac tta ctg tcc 1519 ArgGlu Leu Arg Asp Trp Gly Leu Ser Phe Asp Ser Asn Leu Leu Ser 440 445 450ttc tca gga aga att ttg caa aca gaa aag att cac caa ggt gga aaa 1567 PheSer Gly Arg Ile Leu Gln Thr Glu Lys Ile His Gln Gly Gly Lys 455 460 465aca ttt gat tac aat cca caa ttt gca gat tgg tcc aaa gaa aca aga 1615 ThrPhe Asp Tyr Asn Pro Gln Phe Ala Asp Trp Ser Lys Glu Thr Arg 470 475 480ggt gca cca tta att agt gtt aag cca cta gat aac tgg ctg ttg atc 1663 GlyAla Pro Leu Ile Ser Val Lys Pro Leu Asp Asn Trp Leu Leu Ile 485 490 495tat acg cga aga aat tat gaa gca gcc aat tca ttg ata caa aat cta 1711 TyrThr Arg Arg Asn Tyr Glu Ala Ala Asn Ser Leu Ile Gln Asn Leu 500 505 510515 ttt aaa gtt aca cca gcc atg ggc atg caa atg aga aaa gca ata atg 1759Phe Lys Val Thr Pro Ala Met Gly Met Gln Met Arg Lys Ala Ile Met 520 525530 att gaa gtg gat gac aga act gaa gcc tac tta aga gtc tta cag caa 1807Ile Glu Val Asp Asp Arg Thr Glu Ala Tyr Leu Arg Val Leu Gln Gln 535 540545 aag gtc aca gca gac acc cag ata gtt gtc tgt ctg ttg tca agt aat 1855Lys Val Thr Ala Asp Thr Gln Ile Val Val Cys Leu Leu Ser Ser Asn 550 555560 cgg aag gac aaa tac gat gct att aaa aaa tac ctg tgt aca gat tgc 1903Arg Lys Asp Lys Tyr Asp Ala Ile Lys Lys Tyr Leu Cys Thr Asp Cys 565 570575 cct acc cca agt cag tgt gtg gtg gcc cga acc tta ggc aaa cag caa 1951Pro Thr Pro Ser Gln Cys Val Val Ala Arg Thr Leu Gly Lys Gln Gln 580 585590 595 act gtc atg gcc att gct aca aag att gcc cta cag atg aac tgc aag1999 Thr Val Met Ala Ile Ala Thr Lys Ile Ala Leu Gln Met Asn Cys Lys 600605 610 atg gga gga gag ctc tgg agg gtg gac atc ccc ctg aag ctc gtg atg2047 Met Gly Gly Glu Leu Trp Arg Val Asp Ile Pro Leu Lys Leu Val Met 615620 625 atc gtt ggc atc gat tgt tac cat gac atg aca gct ggg cgg agg tca2095 Ile Val Gly Ile Asp Cys Tyr His Asp Met Thr Ala Gly Arg Arg Ser 630635 640 atc gca gga ttt gtt gcc agc atc aat gaa ggg atg acc cgc tgg ttc2143 Ile Ala Gly Phe Val Ala Ser Ile Asn Glu Gly Met Thr Arg Trp Phe 645650 655 tca cgc tgc ata ttt cag gat aga gga cag gag ctg gta gat ggg ctc2191 Ser Arg Cys Ile Phe Gln Asp Arg Gly Gln Glu Leu Val Asp Gly Leu 660665 670 675 aaa gtc tgc ctg caa gcg gct ctg agg gct tgg aat agc tgc aatgag 2239 Lys Val Cys Leu Gln Ala Ala Leu Arg Ala Trp Asn Ser Cys Asn Glu680 685 690 tac atg ccc agc cgg atc atc gtg tac cgc gat ggc gta gga gacggc 2287 Tyr Met Pro Ser Arg Ile Ile Val Tyr Arg Asp Gly Val Gly Asp Gly695 700 705 cag ctg aaa aca ctg gtg aac tac gaa gtg cca cag ttt ttg gattgt 2335 Gln Leu Lys Thr Leu Val Asn Tyr Glu Val Pro Gln Phe Leu Asp Cys710 715 720 cta aaa tcc att ggt aga ggt tac aac cct aga nta acg gta attgtg 2383 Leu Lys Ser Ile Gly Arg Gly Tyr Asn Pro Arg Xaa Thr Val Ile Val725 730 735 gtg aag aaa aga gtg aac acc aga ttt ttt gct cag tct gga ggaaga 2431 Val Lys Lys Arg Val Asn Thr Arg Phe Phe Ala Gln Ser Gly Gly Arg740 745 750 755 ctt cag aat cca ctt cct gga aca gtt att gat gta gag gttacc aga 2479 Leu Gln Asn Pro Leu Pro Gly Thr Val Ile Asp Val Glu Val ThrArg 760 765 770 cca gaa tgg tat gac ttt ttt atc gtg agc cag gct gtg agaagt ggt 2527 Pro Glu Trp Tyr Asp Phe Phe Ile Val Ser Gln Ala Val Arg SerGly 775 780 785 agt gtt tct ccc aca cat tac aat gtc atc tat gac aac agcggc ctg 2575 Ser Val Ser Pro Thr His Tyr Asn Val Ile Tyr Asp Asn Ser GlyLeu 790 795 800 aag cca gac cac ata cag cgc ttg acc tac aag ctg tgc cacatc tat 2623 Lys Pro Asp His Ile Gln Arg Leu Thr Tyr Lys Leu Cys His IleTyr 805 810 815 tac aac tgg cca ggt gtc att cgt gtt cct gct cct tgc cagtac gcc 2671 Tyr Asn Trp Pro Gly Val Ile Arg Val Pro Ala Pro Cys Gln TyrAla 820 825 830 835 cac aag ctg gct ttt ctt gtt ggc cag agt att cac agagag cca aat 2719 His Lys Leu Ala Phe Leu Val Gly Gln Ser Ile His Arg GluPro Asn 840 845 850 ctg tca ctg tca aac cgc ctt tac tac ctc taacctgcagaagacgatgc 2769 Leu Ser Leu Ser Asn Arg Leu Tyr Tyr Leu 855 860agccgctttt ctttttgaaa tgactttggg atttttttaa gcttttattt actttttttt 2829taactgttat ctttctggat gaaacttggg aaggggatta ggagatctag cattttattt 2889ctagcattgc tattcaccgg cttccttatt ttatatgtaa aaattaagat tttatatttt 2949atcttcttgt ttctcataga tattttgtga gcattttttt gtttattttg aagaaatgtg 3009gataagatac ttggtagtat aaaacagact ctctgagagt atttgaaatg tgtttggaga 3069tttacttaaa cgtactttca ggagtgagca agtcctactt ataaacctat attaacttta 3129tttttgagat acctgttttg aatttaaagg agataagagg cgtaaagtag gatgctcact 3189acaaccatag gtggggtttc agctcatatc ttaaagataa aaggtactat tatataacct 3249atacacaaga tacaggagaa aatatgcttg atttttattt ggcagggggg ctaggttgta 3309tgggagtaaa aaaaacattg aaaattttta aattgtccaa agaaacattt taagactctt 3369taacaaaaaa ggccatgagt aaatctctat attaacatca ctatttattt tgttttggaa 3429ctgggacatg attctatttg ttataaaata aaattgatgt ccc 3472 6 861 PRT Homosapiens misc_feature (76)..(76) The ′Xaa′ at location 76 stands for Ile,Val, or Leu. 6 Met Thr Gly Arg Ala Arg Ala Arg Ala Arg Gly Arg Ala ArgGly Gln 1 5 10 15 Glu Thr Ala Gln Leu Val Gly Ser Thr Ala Ser Gln GlnPro Gly Tyr 20 25 30 Ile Gln Pro Arg Pro Gln Pro Pro Pro Ala Glu Gly GluLeu Phe Gly 35 40 45 Arg Gly Arg Gln Arg Gly Thr Ala Gly Gly Thr Ala LysSer Gln Gly 50 55 60 Leu Gln Ile Ser Ala Gly Phe Gln Glu Leu Ser Xaa AlaGlu Arg Gly 65 70 75 80 Gly Arg Arg Arg Asp Phe His Asp Leu Gly Val AsnThr Arg Gln Asn 85 90 95 Leu Asp His Val Lys Glu Ser Lys Thr Gly Ser SerGly Ile Ile Val 100 105 110 Arg Leu Ser Thr Asn His Phe Arg Leu Thr SerArg Pro Gln Trp Ala 115 120 125 Leu Tyr Gln Tyr His Ile Asp Tyr Asn ProLeu Met Glu Ala Arg Arg 130 135 140 Leu Arg Ser Ala Leu Leu Phe Gln HisGlu Asp Leu Ile Gly Lys Cys 145 150 155 160 His Ala Phe Asp Gly Thr IleLeu Phe Leu Pro Lys Arg Leu Gln Gln 165 170 175 Lys Val Thr Glu Val PheSer Lys Thr Arg Asn Gly Glu Asp Val Arg 180 185 190 Ile Thr Ile Thr LeuThr Asn Glu Leu Pro Pro Thr Ser Pro Thr Cys 195 200 205 Leu Gln Phe TyrAsn Ile Ile Phe Arg Arg Leu Leu Lys Ile Met Asn 210 215 220 Leu Gln GlnIle Gly Arg Asn Tyr Tyr Asn Pro Asn Asp Pro Ile Asp 225 230 235 240 IlePro Ser His Arg Leu Val Ile Trp Pro Gly Phe Thr Thr Ser Ile 245 250 255Leu Gln Tyr Glu Asn Ser Ile Met Leu Cys Thr Asp Val Ser His Lys 260 265270 Val Leu Arg Ser Glu Thr Val Leu Asp Phe Met Phe Asn Phe Tyr His 275280 285 Gln Thr Glu Glu His Lys Phe Gln Glu Gln Val Ser Lys Glu Xaa Ile290 295 300 Gly Leu Val Val Leu Thr Lys Tyr Asn Ile Lys Thr Tyr Arg ValAsp 305 310 315 320 Asp Ile Asp Trp Asp Gln Asn Pro Lys Ser Thr Phe LysLys Ala Asp 325 330 335 Gly Ser Gly Val Ser Phe Leu Glu Tyr Tyr Arg LysGln Tyr Asn Gln 340 345 350 Glu Ile Thr Asp Leu Lys Gln Pro Val Leu ValSer Gln Pro Lys Arg 355 360 365 Arg Arg Gly Pro Gly Gly Thr Leu Pro GlyPro Ala Met Leu Ile Pro 370 375 380 Glu Leu Cys Tyr Leu Thr Gly Leu ThrAsp Lys Met Arg Asn Asp Phe 385 390 395 400 Asn Val Met Lys Asp Leu AlaVal His Thr Arg Leu Thr Pro Glu Gln 405 410 415 Arg Gln Arg Glu Val GlyArg Leu Ile Asp Tyr Ile His Lys Asn Asp 420 425 430 Asn Val Gln Arg GluLeu Arg Asp Trp Gly Leu Ser Phe Asp Ser Asn 435 440 445 Leu Leu Ser PheSer Gly Arg Ile Leu Gln Thr Glu Lys Ile His Gln 450 455 460 Gly Gly LysThr Phe Asp Tyr Asn Pro Gln Phe Ala Asp Trp Ser Lys 465 470 475 480 GluThr Arg Gly Ala Pro Leu Ile Ser Val Lys Pro Leu Asp Asn Trp 485 490 495Leu Leu Ile Tyr Thr Arg Arg Asn Tyr Glu Ala Ala Asn Ser Leu Ile 500 505510 Gln Asn Leu Phe Lys Val Thr Pro Ala Met Gly Met Gln Met Arg Lys 515520 525 Ala Ile Met Ile Glu Val Asp Asp Arg Thr Glu Ala Tyr Leu Arg Val530 535 540 Leu Gln Gln Lys Val Thr Ala Asp Thr Gln Ile Val Val Cys LeuLeu 545 550 555 560 Ser Ser Asn Arg Lys Asp Lys Tyr Asp Ala Ile Lys LysTyr Leu Cys 565 570 575 Thr Asp Cys Pro Thr Pro Ser Gln Cys Val Val AlaArg Thr Leu Gly 580 585 590 Lys Gln Gln Thr Val Met Ala Ile Ala Thr LysIle Ala Leu Gln Met 595 600 605 Asn Cys Lys Met Gly Gly Glu Leu Trp ArgVal Asp Ile Pro Leu Lys 610 615 620 Leu Val Met Ile Val Gly Ile Asp CysTyr His Asp Met Thr Ala Gly 625 630 635 640 Arg Arg Ser Ile Ala Gly PheVal Ala Ser Ile Asn Glu Gly Met Thr 645 650 655 Arg Trp Phe Ser Arg CysIle Phe Gln Asp Arg Gly Gln Glu Leu Val 660 665 670 Asp Gly Leu Lys ValCys Leu Gln Ala Ala Leu Arg Ala Trp Asn Ser 675 680 685 Cys Asn Glu TyrMet Pro Ser Arg Ile Ile Val Tyr Arg Asp Gly Val 690 695 700 Gly Asp GlyGln Leu Lys Thr Leu Val Asn Tyr Glu Val Pro Gln Phe 705 710 715 720 LeuAsp Cys Leu Lys Ser Ile Gly Arg Gly Tyr Asn Pro Arg Xaa Thr 725 730 735Val Ile Val Val Lys Lys Arg Val Asn Thr Arg Phe Phe Ala Gln Ser 740 745750 Gly Gly Arg Leu Gln Asn Pro Leu Pro Gly Thr Val Ile Asp Val Glu 755760 765 Val Thr Arg Pro Glu Trp Tyr Asp Phe Phe Ile Val Ser Gln Ala Val770 775 780 Arg Ser Gly Ser Val Ser Pro Thr His Tyr Asn Val Ile Tyr AspAsn 785 790 795 800 Ser Gly Leu Lys Pro Asp His Ile Gln Arg Leu Thr TyrLys Leu Cys 805 810 815 His Ile Tyr Tyr Asn Trp Pro Gly Val Ile Arg ValPro Ala Pro Cys 820 825 830 Gln Tyr Ala His Lys Leu Ala Phe Leu Val GlyGln Ser Ile His Arg 835 840 845 Glu Pro Asn Leu Ser Leu Ser Asn Arg LeuTyr Tyr Leu 850 855 860 7 42 DNA Artificial Synthesized oligonucleotideprimer 7 gatcatatgg agcaaaagct tattagcgag gaagatctga at 42 8 42 DNAArtificial Synthesized oligonucleotide primer 8 gatcattcag atcttcctcgctaataagct tttgctccat at 42 9 45 DNA Artificial Synthesizedoligonucleotide primer 9 ctagcatatg agcaaaagct tattagcgag gaagatctgaataag 45 10 42 DNA Artificial Synthesized oligonucleotide primer 10ctagcttatt cagatcttcc tcgctaataa gcttttgctc at 42 11 17 DNA ArtificialSynthesized oligonucleotide primer 11 acgataagtt ctgttat 17 12 23 DNAArtificial Synthesized oligonucleotide primer 12 tgcactgcca ggtccttcatcac 23 13 29 DNA Artificial Synthesized oligonucleotide primer 13ggccagtcat tttccagtca gctcaggtg 29 14 20 DNA Artificial Synthesizedoligonucleotide primer 14 atggggtctt ttcttgctca 20 15 20 DNA ArtificialSynthesized oligonucleotide primer 15 tgcccattaa catcaccatc 20 16 24 DNAArtificial Synthesized oligonucleotide primer 16 tgatttgggg acttattttagagc 24 17 23 DNA Artificial Synthesized oligonucleotide primer 17acttaccttg tgacttggat gtg 23 18 24 DNA Artificial Synthesizedoligonucleotide primer 18 ttgaaaagca ttgaacacca taag 24 19 24 DNAArtificial Synthesized oligonucleotide primer 19 gataaccacc gccctgcctttcac 24 20 20 DNA Artificial Synthesized oligonucleotide primer 20tgcccattaa catcaccatc 20 21 4 PRT Artificial linker peptide 21 Pro ProArg Gln 1

1. An isolated and purified biologically active piwi family polypeptidecomprising: (a) a polypeptide encoded by a nucleic acid sequence as setforth in any of SEQ ID NOs:1, 3 and 5; (b) a polypeptide encoded by anucleic acid having at least about 75% or greater homology to a DNAsequence as set forth in any of SEQ ID NOs:1, 3 and 5; (c) a polypeptideencoded by a nucleic acid capable of hybridizing under stringentconditions to a nucleic acid comprising a sequence or the complement ofa sequence as set forth in any of SEQ ID NOs:1, 3 and 5; (d) apolypeptide which is a biologically functional equivalent of a peptideas set forth in any of SEQ ID NOs:2, 4 and 6; (e) a polypeptide which isimmunologically cross-reactive with antibodies which are immunologicallyreactive with peptides encoded by the nucleic acid sequences as setforth in any of SEQ ID NOs:2, 4 and 6; (f) a polypeptide encoded by anucleic acid molecule capable of hybridizing to a nucleic acid moleculehaving the sequence of any of SEQ ID NOs:1, 3 and 5, or fragments orvariants or complementary sequences thereof, under high stringencyconditions; or (g) a polypeptide comprising a portion of a polypeptideof a), b), c), d), e), or f).
 2. The polypeptide of claim 1, wherein thepolypeptide comprises a vertebrate piwi family polypeptide.
 3. Thepolypeptide of claim 2, wherein the polypeptide comprises a human piwifamily polypeptide.
 4. The polypeptide of claim 1, wherein the piwifamily comprises an amino acid sequence as set forth in any of SEQ IDNOs:2, 4 and
 6. 5. The polypeptide of claim 1, modified to be indetectably labeled form.
 6. An isolated and purified antibody capable ofspecifically binding to the polypeptide of claim
 1. 7. The antibody ofclaim 6 which is a monoclonal antibody.
 8. The antibody of claim 6 whichis a polyclonal antibody.
 9. A hybridoma cell line which produces themonoclonal antibody of claim
 8. 10. An isolated and purified antibodycapable of neutralizing the biological activity of the polypeptide ofclaim
 1. 11. The antibody of claim 10 which is a monoclonal antibody.12. The antibody of claim 10 which is a polyclonal antibody.
 13. Ahybridoma cell line which produces the monoclonal antibody of claim 11.14. An isolated and purified polynucleic acid encoding a biologicallyactive piwi family polypeptide of claim
 1. 15-29. (Canceled).
 30. Amethod of detecting in a sample an RNA that encodes the piwi familypolypeptide encoded by the nucleic acid of claim 14, said methodcomprising the steps of: (a) contacting said sample under hybridizingconditions with the polynucleic acid of claim 14 to form a duplex; and(b) detecting the presence of said duplex.
 31. A method of producing anantibody immunoreactive with a piwi family polypeptide, the methodcomprising steps of: (a) transfecting a recombinant host cell with the apolynucleic acid of claim 14, which encodes a piwi family polypeptide;(b) culturing the host cell under conditions sufficient for expressionof the polypeptide; (c) recovering the polypeptide; and (d) preparingthe antibody to the polypeptide.
 32. The method of claim 31, wherein thepolypeptide comprises a polypeptide as essentially set forth in any ofSEQ ID NOs:2, 4 and
 6. 33. The method of claim 31, wherein thepolynucleic acid comprises a polynucleic acid sequence as essentiallyset forth in any of SEQ ID NOs:1, 3 and
 5. 34. An antibody produced bythe method of claim
 31. 35. A method of detecting a piwi familypolypeptide, the method comprising immunoreacting the polypeptide withan antibody prepared according the method of claim 31 to form anantibody-polypeptide conjugate; and detecting the conjugate.
 36. Amethod of detecting a messenger RNA transcript that encodes a piwifamily polypeptide, the method comprising the steps of hybridizing themessenger RNA transcript with the polynucleic acid of claim 14 to form aduplex; and detecting the duplex.
 37. A method of detecting a DNAmolecule that encodes a piwi family polypeptide, the method comprisingthe steps of hybridizing DNA molecules with the polynucleic acid ofclaim 14 to form a duplex; and detecting the duplex.
 38. An assay kitfor detecting the presence of a piwi family polypeptide in a biologicalsample, the kit comprising a first container containing a first antibodycapable of immunoreacting with a piwi family polypeptide of claim 1,wherein the first antibody is present in an amount sufficient to performat least one assay.
 39. The assay kit of claim 38, further comprising asecond container containing a second antibody that immunoreacts with thefirst antibody.
 40. The assay kit of claim 39, wherein the firstantibody and the second antibody comprise monoclonal antibodies.
 41. Theassay kit of claim 39, wherein the first antibody is affixed to a solidsupport.
 42. The assay kit of claim 39, wherein the first and secondantibodies each comprise an indicator.
 43. The assay kit of claim 42,wherein the indicator is a radioactive label, a fluorescent label or anenzyme.
 44. An assay kit for detecting the presence, in biologicalsamples, of a piwi family polypeptide, the kit comprising a firstcontainer that contains a polynucleic acid identical or complimentary toa segment of at least ten contiguous nucleotide bases of the polynucleicacid of claim
 14. 45. An assay kit for detecting the presence, in abiological sample, of an antibody immunoreactive with a piwi familypolypeptide, the kit comprising a first container containing a piwifamily polypeptide of claim 1 that immunoreacts with the antibody, withthe polypeptide present in an amount sufficient to perform at least oneassay.
 46. A method of screening candidate substances for an ability tomodulate piwi family biological activity, the method comprising thesteps of: (a) establishing replicate test and control samples thatcomprise a biologically active piwi family polypeptide; (b)administering a candidate substance to the test sample but not thecontrol sample; (c) measuring piwi family biological activity in thetest and the control samples; and (d) determining that the candidatesubstance modulates piwi family biological activity if the piwi familybiological activity measured for the test sample is greater or less thanthe piwi family biological activity measured for the control sample. 47.The method of claim 46, wherein the candidate substance is furthercharacterized as a candidate polypeptide, and further comprising thestep of purifying and isolating a gene encoding the candidatepolypeptide.
 48. The method of claim 46, wherein the replicate test andcontrol samples further comprise a cell that expresses a biologicallyactive vertebrate piwi family polypeptide.
 49. (Canceled).
 50. A methodof modulating piwi family polypeptide activity in a subject, the methodcomprising the step of administering to the subject an effective amountof a substance capable of modulating the piwi family polypeptideactivity in the subject, whereby modulation of the piwi familypolypeptide activity is accomplished.
 51. The method of claim 50,wherein the step of administering further comprises administering aneffective amount of a substance that modulates expression of a piwifamily-encoding polynucleic acid in the subject.
 52. The method of claim51, wherein the substance that modulates expression of a piwifamily-encoding polynucleic acid comprises an antisense oligonucleotide.53. The method of claim 50, where the substance that modulates the piwifamily activity comprises an anti-piwi family antibody.
 54. The methodof claim 53, where the anti-piwi family antibody comprises a monoclonalantibody.
 55. The method of claim 50, wherein the piwi family activitycomprises modulating cell growth, proliferation, division andcombinations thereof, and wherein the step of administering comprisesadministering to the subject an effective piwi family-modulating amountof a piwi family-modulating substance.
 56. The method of claim 50,wherein the subject is a vertebrate.
 57. The method of claim 56, whereinthe vertebrate is a warm-blooded vertebrate.
 58. A method of treating asubject suffering from a disorder associated with piwi family biologicalactivity, the method comprising: administering to the subject aneffective amount of a piwi family biological activity-modulatingsubstance, whereby treatment of the disorder is accomplished.
 59. Themethod of claim 58, wherein the step of administering further comprisesadministering an effective amount of a substance that modulatesexpression of a piwi family-encoding polynucleic acid in the subject.60. The method of claim 59, wherein the substance that modulatesexpression of a piwi family-encoding polynucleic acid comprises anantisense oligonucleotide.
 61. The method of claim 58, wherein thesubstance capable of modulating piwi family activity in the subjectcomprises an anti-piwi family antibody.
 62. The method of claim 61,wherein the anti-piwi family antibody comprises a monoclonal antibody.63. A method of treating a subject suffering from a disorder associatedwith piwi-mediated biological activity, the method comprisingadministering to the subject a therapeutic composition which comprises abiologically active piwi family polypeptide, whereby treatment ofdisorder associated with piwi-mediated biological activity in thesubject is accomplished.
 64. The method of claim 63, wherein thetherapeutic composition comprises a biologically active piwi familypolypeptide and a pharmaceutically acceptable carrier.
 65. The method ofclaim 63, wherein the piwi-mediated biological activity comprisesmodulation of stem cell growth, proliferation, division and combinationsthereof, modulation of primordial germ cell proliferation, andcombinations thereof; and the piwi family polypeptide is administered inan effective amount.
 66. A method for modulating piwi-mediatedbiological activity in a cell, the method comprising: (a) delivering tothe cell an effective amount of a DNA molecule comprising apolynucleotide that encodes a biologically active piwi familypolypeptide; and (b) maintaining the cell under conditions sufficientfor expression of said polypeptide.
 67. The method of claim 66, whereinthe polypeptide comprises a polypeptide as essentially set forth in anyof SEQ ID NOs:2, 4 and
 6. 68. The method of claim 67, wherein thepolypeptide is encoded by a polynucleic acid as essentially set forth inany of SEQ ID NOs:1, 3 and
 5. 69. A method of culturing a primitivecell, the method comprising: (a) providing a culture comprising aprimitive cell; and (b) delivering to the primitive cell a piwi familygene product, whereby growth of the primitive cell in a culture isaccomplished.
 70. The method of claim 69, wherein the primitive cell ismaintained in the culture for a time sufficient to produce a sustainedcell culture.
 71. The method of claim 69, wherein the piwi family geneproduct is delivered to the primitive cell by delivering to theprimitive cell an effective amount of a polynucleotide that encodes abiologically active piwi family polypeptide; and maintaining theprimitive cell under conditions sufficient for expression of thepolypeptide.
 72. The method of claim 69, wherein the piwi family geneproduct is delivered to the cell by administration of a biologicallyactive piwi family polypeptide to the culture.
 73. The method of claim69, further comprising: (a) collecting primitive cells; (b) depositingthe primitive cells in contact with a feeder matrix, the feeder matrixcomprising a piwi family gene product; and (c) culturing the primitivecells on the feeder matrix in the presence of media for a timesufficient to produce a sustained primitive cell culture.
 74. The methodof claim 69, wherein the feeder matrix comprises a recombinant cellcomprising a polynucleic acid encoding a biologically active piwi familypolypeptide.
 75. The method of claim 69, further comprising establishinga monolayer of cells in the feeder matrix.
 76. A transgenic or chimericnon-human animal having incorporated into its genome a polynucleic acidencoding a biologically active piwi family polypeptide, the polynucleicacid being present in said genome in a copy number effective to conferexpression in the animal of the piwi family polypeptide.
 77. Thetransgenic or chimeric non-human animal of claim 76, wherein saidpolynucleic acid comprises a human piwi family-encoding segment.
 78. Thetransgenic or chimeric non-human animal of claim 76, wherein theexpression of the piwi family polypeptide is conferred in reproductivetissue of the animal.
 79. The transgenic or chimeric non-human animal ofclaim 76, wherein the expression of the piwi family polypeptide confersan increased number of germline stem cells or primordial germ cells inthe animal as compared to a normal animal.
 80. The transgenic orchimeric non-human animal of claim 76, further comprising an additionalpolynucleic acid encoding a predetermined biologically activepolypeptide, the polynucleic acid being present in said genome in a copynumber effective to confer expression in the animal of the polypeptide.81. A method of altering the phenotype of an embryonic animal, themethod comprising: (a) providing a recombinant primitive cell comprisinga polynucleic acid encoding a biologically active piwi familypolypeptide; (b) transfecting the recombinant primitive cell with apolynucleic acid encoding a predetermined biologically activepolypeptide; (c) transferring the transfected primitive cells into anembryo to confer expression of the predetermined biologically activepolypeptide animal of the polypeptide, whereby the phenotype of theembryo is altered.
 82. The method of claim 81, wherein the animal is abird, and the transfected primitive cell is transferred into an eggcontaining an embryonic bird.
 83. The method of claim 81, wherein thealtered phenotype comprises a change in protein expression, a change ingrowth rate, feed efficiency, a change in stem cell count, a change inprimordial germ cell count, disease resistance, or a combinationthereof.
 84. The method of claim 81, wherein the embryonic animal ispermitted to develop into a fetus or animal.
 85. A method of recoveringa protein from a transgenic or chimeric non-human animal, the methodcomprising: (a) providing a transgenic or chimeric non-human animalhaving incorporated into its genome a polynucleic acid encoding abiologically active piwi family polypeptide, the polynucleic acid beingpresent in said genome in a copy number effective to confer expressionin the animal of the piwi family polypeptide; and (b) recovering thebiologically active piwi family polypeptide from the animal provided instep (a).
 86. The method of claim 85, wherein the transgenic or chimericnon-human animal provided in step (a) further comprises anotherpolynucleic acid incorporated into its genome, the polynucleic acidencoding a predetermined biologically active polypeptide, thepolynucleic acid being present in said genome in a copy number effectiveto confer expression in the animal of the predetermined polypeptide; andwherein the predetermined biologically active polypeptide is recoveredin addition to or instead of the biologically active piwi familypolypeptide.
 87. The method of claim 85 or 86, wherein the transgenic orchimeric non-human animal provided in step (a) further comprises anavian, and the polypeptide is recovered from an egg produced by theavian.
 88. A pharmaceutical composition comprising an isolated andpurified biologically active piwi family polypeptide and apharmaceutically acceptable carrier.
 89. The pharmaceutical compositionof claim 88, wherein the polypeptide comprises a polypeptide asessentially set forth in any of SEQ ID NOs:2, 4 and
 6. 90. Thepharmaceutical composition of claim 88, wherein the polypeptide isencoded by a polynucleic acid sequence as essentially set forth in anyof SEQ ID NOs:1, 3 and
 5. 91. The pharmaceutical composition of claim88, further comprising a cell expressing the polypeptide.
 92. Apharmaceutical composition comprising an isolated and purifiedpolynucleic acid encoding a biologically active piwi family polypeptideand a pharmaceutically acceptable carrier.
 93. The pharmaceuticalcomposition of claim 92, wherein the encoded polypeptide comprises anamino acid sequence as essentially set forth in any of SEQ ID NOs:2, 4and
 6. 94. The pharmaceutical composition of claim 92, furthercomprising a piwi family-encoding polynucleic acid sequence asessentially set forth in any of SEQ ID NOs:1, 3 and
 5. 95. Thepharmaceutical composition of claim 92, wherein the polynucleic acid isa DNA segment.
 96. The pharmaceutical composition of claim 92, whereinthe polynucleic acid is positioned under the control of a promoter. 97.The pharmaceutical composition of claim 92, wherein the polynucleic acidfurther comprises a recombinant vector.
 98. The pharmaceuticalcomposition of claim 92, further comprising a cell expressing theencoded polypeptide.